Hafnia Kangkung.docx

BAB I PENDAHULUAN

1.1 Latar belakang

Hafnia Alvei merupakan sosok salah satu penyebab penyakit pencernaan. Bahkan sekarang ini di Indonesia sering terjadi penyakit pencernaan yang disebabkan oleh organisme yang satu ini.

Penyakit pencenaan akibat organisme ini telah mendapat perhatian klinis oleh para ahli. Dulu organisme ini tidak mendapat perhatian oleh para ahli dikarenakan penyakit yang ditimbulkan belum menjangkiti banyak orang. Namun sekarang ini masyarakat Indonesia mulai “akrab” dengan penyakit yang ditimbulkan oleh organisme ini, sehingga diperlukan ada penjelasan yang lebih lengkap tentang organisme ini. Ketika era globalisasi menyebabkan informasi semakin mudah diperoleh, negara berkembang dapat segera meniru kebiasaan negara barat yang dianggap cermin pola hidup modern. Masyarakat Indonesia meniru kebiasaan, pola makan, busana, dan berbagai macam hal mengenai orang barat tetapi orang Indonesia sendiri belum meniru mengenai kebersihan lingkungan. Hal ini menyebabkan penyakit yang disebabkan oleh organisme ini semakin merajalela.

1.2 Permasalahan

Permasalahan yang dangkat dalam penulisan makalah ini ialah: 1. Apa itu bakteri Hafnia alvei? 2. Apa ciri- ciri bakteri Hafnia alvei? 3. Apa penyakit yang diakibatkan oleh bakteri Hafnia alvei?

H. Alvei

Page 1

1.3. Tujuan Tujuan pembuatan karya tulis ini ialah : 1. Ingin mengetahui dengan baik mengenai apa itu bakteri Hafnia alvei. 2. Menjelaskan ciri – ciri dari bakteri Hafnia alvei. 3. Mengetahui contoh kasus penyakit yang diakibatkan oleh bakteri Hafnia alvei.

1.4. Manfaat Manfaat yang diperoleh dari pembuatan karya tulis ini ialah : 1. Dapat mengerti dengan baik tentang bakteri Hafnia alvei. 2. Dapat menentukan jenis bakteri Hafnia alvei berdasarkan ciri – ciri yang telah diketahui. 3. Dapat mengetahui contoh kasus penyakit yang disebabkan oleh bakteri Hafnia alvei.

H. Alvei

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BAB II PEMBAHASAN

2.1. Pengertian

Kingdom: Bacteria Phylum:

Proteobacteria

Class:

Gammaproteobacteria

Order:

Enterobacteriales

Family:

Enterobacteriaceae

Genus:

Hafnia Møller, 1954

Hafnia adalah genus dari Enterobacteriaceae keluarga yang hanya spesies adalah Gram-negatif , fakultatif anaerob , berbentuk batang bakteri Hafnia alvei. H. alvei adalah komensal pada saluran pencernaan manusia dan biasanya tidak patogen , tetapi dapat menyebabkan penyakit pada immunocompromised pasien.

Hal

ini

sering

resisten

terhadap

antibiotik

ganda,

termasuk

aminopenicillins. Cara-Hafnia hanya Hafnia alvei merupakan penghuni berbahaya dari usus dan merupakan bagian dari alam flora usus manusia. Hanya jarang adalah infeksi bakteri di dalam individu dikompromikan, seperti transplantasi organ, atau karena

H. Alvei

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alasan lain yang terlibat pasien immunocompromised dan dalam darah , nanah , dahak , atau urin untuk menemukan. Terapi seharusnya tidak akut infeksi yang mengancam jiwa dalam antibiogram orientasi inti terisolasi. Selama ini tidak tersedia,

terapi

dengan

fluoroquinolone atau

cephalosporin

dengan antibiotik

generasi

ketiga,

kelompok

II

Pseudomonas-acting beta-laktam,

mungkin mulai dalam kombinasi dengan antibiotik aminoglikosida. Patogen tahan terhadap banyak antibiotik, termasuk terhadap aminopenicillins.

H. alvei juga terdapat di berbagai mamalia, ikan, burung, tanah, air, dan sejumlah makanan. H. alvei memiliki mekanisme virulensi beberapa yang berbeda, yang mirip atau identik dengan gram negatif enteropatogen lainnya. Pada manusia, H. alvei merupakan penyebab sejumlah penyakit, termasuk pneumonia, meningitis, abses, dan septicaemia. Patogenesis: Strain mungkin terisolasi dari kotoran manusia dan hewan lainnya. Mereka juga dapat ditemukan dalam limbah, air tanah, dan produk susu. Ini merupakan

H. Alvei

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bug lingkungan, sebagian besar.Beberapa strain membawa gen penyandi kemampuan untuk menyebabkan "melampirkan dan merendahkan" lesi sel usus, mirip dengan EPEC (enteropathic E. coli). Ini Gram-negatif bacillus enterik dan orofaringeal biasanya non-patogen. Hal ini assocaited dengan penyakit yang mendasari kronis (dan mungkin terjadi pada isolat pasien 'endotracheally diintubasi), dan dapat menjadi penyebab pneumonia dalam pengaturan masyarakat atau rumah sakit. Isolat biasanya menampilkanresistensi terhadap antibiotik konvensional

termasuk

sefalosporin

dan penisilin. Meskipun

jarang, H.alvei mungkin patogen potensial dalam pasien dengan penyakit yang mendasari kronis.

Kerentanan: Hafnia alvei sering resisten terhadap ampisilin dan sefalosporin generasi 1 dan 2.Organisme ini umumnya menunjukkan kerentanan untuk kuinolon, aminoglikosida,

kloramfenikol,

kotrimoksazol, sefepim,

aztreonam

dan

carbapenems, sedangkan kerentanan terhadap tetrasiklin adalah variabel.

Perlawanan: Hafnia alvei memiliki kelompok Bush 1, Ambler kelas C betalactamase yang kromosomnya dikodekan dan mungkin diinduksi atau tingkat tinggi secara konstitusional dinyatakan. 2.2. Ciri – ciri bakteri Hafnia alvei

Morfologi :

Pewarnaan

: Gram-negatif

Morfologi

: Lurus batang, 1,0 um dan diameter 2,0-5,0 um panjang.

Motilitas

: Motil oleh flagela peritrichous pada 30 `C, namun strain nonmotile mungkin terjadi

H. Alvei

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Khusus

Tidak dienkapsulasi

Colonial : Permukaan

Tumbuh mudah pada media biasa. Koloni pada agar nutrien

padat

umumnya 2-4 mm, halus, lembab, tembus, dan abu-abu dengan permukaan mengkilap dan tepi seluruh

Fisiologis : Tropisme

Chemoorganotrophic. Mayoritas strain memanfaatkan sitrat, asetat dan malonat sebagai sumber karbon tunggal setelah 3-4 hari inkubasi

Oksigen

Fakultatif anaerob, memiliki keduanya pernapasan dan jenis fermentasi metabolisme

Produk

Nitrat direduksi menjadi nitrit. H2S tidak diproduksi dalam gagang besi agar Kligler .. Alginat tidak dimanfaatkan. Pektat tidak membusuk. Fenilalanin deaminate tidak diproduksi

Enzim

Oksidase-negatif. Katalase-positif gelatinase, lipase, dan deoxyribonuclease tidak diproduksi tes dekarboksilase ornithine Lisin dan positif, namun tes dihydrolase arginin merupakan Glukosa negatif difermentasi dengan produksi asam dan gas. Asam tidak diproduksi dari D-sorbitol, raffinose, melibiose, D-adonitol dan myo-inositol. Tes metil merah biasanya positif pada 35 `C dan negatif pada 22` C. ... Acetylmethylcarbinol biasanya diproduksi dari glukosa pada 2228 `C tetapi tidak dapat diproduksi di 35` C.

Lingkungan : Habitat

Terjadi dalam tinja manusia dan hewan lainnya termasuk burung, juga terjadi pada produk-produk limbah, tanah, air dan susu

Genome : G+ C% Mol

48-49 (Tm)

Apa yang tampak seperti pada agar-agar: 

H. Alvei

Colony Seluruh Putaran; punctiform

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

Margin Halus, seluruh



Elevation Convex



Warna Off-putih; mengkilap

Biokimia tes: 

Oksidase negatif



Katalase positif



Indole negatif



Simmons Citrate negatif



Metil Merah positif



Positif Voges-Proskauer



Lisin dekarboksilase positif



H 2 S produksi negatif



Urease negatif



Pengurangan Nitrat positif



Fenilalanin deaminase negatif

Fakta Menarik: 

Dapat terjadi pada tinja manusia dan hewan lainnya, termasuk burung, dan limbah, air tanah, dan produk susu.



Sebuah patogen oportunistik bagi manusia, biasanya dalam darah, urin, atau infeksi luka.

H. Alvei

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2.3 Contoh kasus penyakit yang disebabkan oleh bakteri Hafnia alvei Contoh kasus I : Sebuah tinjauan literatur dan faktor-faktor predisposisi mungkin untuk Hafnia peritonitis dibahas. Seorang wanita 73 tahun menderita stadium akhir gagal ginjal karena nefropati diabetes dan dimulai pada PD rawat jalan terus menerus (CAPD) pada tahun 2005. Dia disajikan dengan demam, sakit perut, dan keruh dialisat ke Rumah Sakit Queen Mary. Pasien ini mengalami kekurangan gizi karena makan, mulut yang buruk dan albumin serum nya hanya 25 g / L pada masuk. Jumlah sel efluen adalah sampai dengan 3230 × 10 6 / L pada presentasi dan diagnosis CAPD peritonitis dibuat. Dia mulai intraperitoneal cefazolin dan tobramycin tetapi dengan respon yang tidak memuaskan. The Gram awal noda cairan peritoneal menunjukkan gram negatif batang dan rezim antibiotik beralih ke ceftazidime intraperitoneal dan amikasin 4 hari setelah masuk. Pasien menanggapi secara klinis dan cairan peritoneal selanjutnya tumbuh Hafnia alvei sensitif terhadap cefuroxime dan gentamisin. Sebagai tambahan terhadap antibiotik intraperitoneal, pasien ini juga menerima nutrisi orangtua dalam pandangan makan yang buruk lisan dan status gizi. Setelah 2 minggu ceftazidime intraperitoneal dan amikasin, dia peritonitis diatasi secara tuntas dan dia cocok untuk debit. Tidak ada kambuh peritonitis pada tindak lanjut. Hafnia alvei milik Enterobacteriaceae keluarga dan merupakan bagian dari flora usus manusia. Setelah dianggap sebagai komensal sederhana dari saluran pencernaan, ada peningkatan bukti untuk menyarankan H. alvei adalah bakteri langka tapi signifikan yang dapat menyebabkan infeksi oportunistik pada manusia. Selain deskripsi dari gastroenteritis yang disebabkan oleh spesies ini, ada laporan berbagai kasus keterlibatan ekstraintestinal infeksi Hafnia. Infeksi saluran

pernapasan

dan

bakteremia

adalah

manifestasi

ekstraintestinal

terkemuka H. alvei sebagai patogen. Kasus lain yang dilaporkan termasuk infeksi luka, abses hati, dan endophthalmitis. Meskipun ada laporan kasus pada spontan peritonitis bakteri pada pasien dengan mesothelioma peritoneal, kasus kami adalah laporan pertama dariH. alvei PD-terkait peritonitis. Penting untuk dicatat bahwa Hafnia ekstraintestinal infeksi nosokomial dan biasanya berhubungan

H. Alvei

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dengan penyakit yang mendasari atau faktor predisposisi. Laporan kasus sebelumnya telah digambarkan infeksi HIV, penyakit hati, transplantasi organ padat, dan keganasan terkait dengan infeksi ekstraintestinal oleh H. Alvei. Dalam pasien kami, faktor risiko kemungkinan adalah diabetes mellitus, gizi buruk, dan gagal ginjal, yang diberikan padanya rentan terhadap infeksi oportunistik. Mobley juga menggambarkan sebuah kasus Hafnia septicemia pada pasien dengan diabetes mellitus. Presentasi klinis pasien kami mirip dengan PD-terkait peritonitis yang disebabkan oleh organisme lain, dengan demam, sakit perut, dan keruh

limbah. Pengobatan

infeksi

ini

tidak

berbeda

dari

peritonitis

biasanya. Hafnia biasanya rentan terhadap carbapenems, monobactams, kuinolon, dan

aminoglikosida. Meskipun

beberapa H.strain alvei memproduksi

kedua

cephalosporinase tingkat rendah inducible (ceftazidime rentan) dan tinggi-tingkat aktivitas cephalosporinase konstitutif yang resisten terhadap ceftazidime, strain diisolasi

dari

pasien

kami

adalah

rentan

terhadap

sefalosporin

dan

aminoglikosida. Pengalaman kami menunjukkan bahwa H. alvei peritonitis dapat berhasil diobati dengan antibiotik yang tepat intraperitoneal dan penghapusan kateter Tenckhoff mungkin tidak diperlukan. Tindakan adjunctive lainnya termasuk perbaikan status gizi dengan makan parenteral. Mengingat asal pencernaan nya, divertikulitis sigmoid telah dilaporkan dikaitkan dengan Hafnia bakteremia. Dengan demikian kami percaya bahwa hal itu juga mungkin layaksaat untuk mencari patologi usus pada pasien dengan Hafnia peritonitis.

Contoh kasus II : Seorang pasien 9 tahun wanita dengan anemia Fanconi dirawat di Rumah Sakit Santo Spirito, Pescara, Italia, pada tanggal 4 Oktober 2007, dalam rangka untuk menerima transplantasi sel induk haematopoietic (menggunakan sel darah tali pusat yang tidak terkait). Profilaksis dengan kotrimoksazol diberikan 4 sampai 15 Oktober 2007. Juga, standar imunosupresif pra-transplantasi pengobatan dengan siklosporin dan dosis rendah kortikosteroid dimulai pada tanggal 13 Oktober 2007, dan pasien masih menerima pengobatan ini pada 1 Februari 2008. Sampel tinja dikultur dua kali seminggu, dan menunjukkan adanya flora komensal

H. Alvei

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enterik (sebagian besar diwakili olehEscherichia coli). Pada tanggal 30 Oktober 2007, Hafnia alvei muncul dalam sampel tinja untuk pertama kalinya, saat jumlah bakteri yang tinggi (> 200 cfu per piring) dan sebagai pertumbuhan murni berat. Identifikasi pada kepastian 99% dibuat menggunakan sistem Vitek2 dan dikonfirmasi oleh sistem API mini (kedua instrumen oleh bioMérieux). Isolat yang menunjukkan kerentanan terhadap cefoxitin (MIC ≤ 4 mg ml -1), sefepim (MIC ≤ 1 mg ml -1), imipenem (MIC ≤ 1 mg ml -1), meropenem (MIC ≤ 0,25 mg ml -1), amikasin (MIC ≤ 2 mg ml -1), siprofloksasin (MIC ≤ 0,25 mg ml -1), levofloxacin (MIC ≤ 0,25 mg ml -1) dan tetrasiklin (MIC 4 mg ml -1), namun resistensi terhadap ampisilin (MIC ≥ 32 mg ml -1), piperasilin (MIC ≥ 256 mg ml -1), amoksisilin / klavulanat (MIC ≥ 32 mg ml -1), ampisilin / sulbaktam (MIC ≥ 32 mg ml -1), piperasilin / Tazobactam (MIC ≥ 128 mg ml -1), ceftazidime (MIC ≥ 64 mg ml -1) dan kotrimoksazol (MIC ≥ 320 mg ml -1). MIC ditentukan oleh sistem Vitek2. Pada tanggal 18 November 2007, pasien mengalami diare akut dan kuat kram-jenis sakit perut. Piring budaya dari kotoran tumbuh H. alvei (> 200 cfu per piring) sebagai organisme tunggal lagi. Tidak ada organisme lain (bakteri, jamur, virus atau parasit) dari patogenisitas usus diketahui terdeteksi. Juga, Clostridium difficile tidak ditemukan dalam budaya, dan C. difficile A / B racun tidak diungkapkan oleh tes immunoenzymic kami melakukan (C. difficile panel; Biosite). H. alvei sebagai agen penyebab kemungkinan untuk enteritis itu dianggap tidak pasti, karena ekspresi enteropathogenicity oleh organisme ini masih kontroversial saat ini ( Janda & Abbott, 2006 ). Juga, korupsi akut yang khas dibandingkan penyakit host (GVHD) lesi kulit muncul, sehingga GVHD enterik dianggap sebagai penyebab paling mungkin. Sebagai pasien juga menderita bakteremia Staphylococcus aureus (data tidak ditampilkan) selama periode rawat inap, ceftazidime, amikasin dan teicoplanin yang dimulai pada tanggal 24 November 2007 sebagai terapi kombinasi parenteral (10 hari terapi). Antimikroba menyebabkan resolusi bakteremia, sedangkan nyeri perut dan diare bertahan. Juga, meskipun kerentanan didokumentasikan H. alvei ke amikasin, spesies dikultur sebagai organisme tunggal dari sampel tinja baru (> 200 cfu per piring). Akhirnya, GVHD enterik didokumentasikan oleh endoskopi dan biopsi usus pada tanggal 3 Desember 2007. Dosis rendah kortikosteroid diubah menjadi

H. Alvei

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kortikosteroid dosis tinggi, dan anti-limfosit mAbs yang dimulai, yang mengarah ke lega bertahap gejala dalam waktu 1 minggu. Pada tanggal 17 Desember 2007, kortikosteroid diubah kembali ke pengobatan dosis rendah, mAbs sementara dihentikan. Menariknya, antimikroba telah berhenti sudah, pada tanggal 4 Desember 2007, sedangkanH. alvei menghilang dari sampel tinja dalam waktu 10 hari dari resolusi klinis (budaya positif terakhir yang diperoleh pada tanggal 20 Desember 2007). H. alvei adalah patogen manusia langka. Beberapa kasus gastroenteritis dilaporkan dalam literatur telah diduga karena organisme ini, namun perannya sebagai agen infeksi enterik masih tak menentu ( Gunthard & Pennekamp, 1996 , Janda & Abbott, 2006 ). Isolasi dari sekresi pernafasan lebih umum, walaupun sebagian besar isolat dari saluran udara tampaknya tidak signifikan secara klinis, laporan sporadis pneumonia, abses paru dan bronkopneumonia yang kemungkinan besar karena H. alvei telah dijelaskan ( Gunthard & Pennekamp, 1996 , Janda & Abbott, 2006 ). Organisme ini kadang-kadang ditemukan di saluran kemih, biasanya sebagai komensal, meskipun dalam beberapa kasus itu dianggap signifikan secara klinis ( Gunthard & Pennekamp, 1996 , Janda & Abbott, 2006 ). Langka aliran darah di mana infeksi H. alveidiisolasi dari kultur darah telah dilaporkan. Bacteraemias kebanyakan masyarakat diperoleh, dan waktu dari kultur darah positif pertama biasanya berkisar antara 1 sampai 41 hari setelah rawat inap. Dalam beberapa kasus, organisme telah diisolasi dari darah dan abses hati, cairan pankreas pseudokista, cairan pleura, dan kateter vena sentral, pada saat yang sama, meskipun sumber bacteraemias sebagian besar tetap tidak diketahui, asal utama dari infeksi aliran darah dianggap menjadi saluran pernapasan atau usus ( Gunthard & Pennekamp, 1996 , Janda & Abbott, 2006 , Liu et al, 2007. ; .Rodriguez-Guardado et al, 2006 ). Neonatal infeksi yang terkait dengan diamH. alvei kereta vagina oleh ibu dan kasus meningitis pada pasien 1-tahun telah dilaporkan juga. Sangat sedikit laporan yang ada dalam literatur mengenai kolonisasi luka oleh organisme ini, dan kami menemukan hanya dua laporan mengenai H. alvei endophthalmitis ( Gunthard & Pennekamp, 1996 ,Janda & Abbott, 2006 ). Beberapa kasus isolasi ini organisme dari abses, dan satu dari pasien dengan septic arthritis, diketahui, namun perannya sebagai patogen tidak

H. Alvei

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menentu sejak pulih sebagai bagian dari flora bakteri campuran ( Gunthard & Pennekamp, 1996 ; Janda & Abbott, 2006 ). Akhirnya, dua kasus kolesistitis, sebuah laporan dari bakteri spontan peritonitis dan kasus endokarditis telah dijelaskan ( Hazouard et al, 2006. , Janda & Abbott, 2006 ; .Loulergue et al, 2007). Menariknya, wabah sindrom uremik hemolitik kemungkinan telah dikutip oleh Janda dan rekan, di mana H. alvei regangan menghasilkan racun sel Vero cytolytic aktif diisolasi dari faeces ( Crandall et al., 2006 ). H. alvei dikenal menjadi patogen nosokomial biasa, namun sedikit yang telah ditulis mengenai perannya sebagai agen oportunistik infeksi pada pasien immunocompromised. Khususnya, tidak ada data yang ada pada saat ini sekitar GVHD sebagai faktor risiko yang mungkin untuk memperoleh H. alveikolonisasi dan / atau infeksi. GVHD akut biasanya terjadi setelah transplantasi sel induk alogenik, dan merupakan reaksi donor yang diturunkan sel T melawan tuan rumah, hati kulit atau usus. Penyakit enterik dicurigai ketika pasien mengembangkan tanda-tanda atau gejala-gejala seperti kram-jenis sakit perut, diare, mual dan muntah. Namun, karena ini adalah gejala non-spesifik, endoskopi, biopsi dan konfirmasi histologis, seperti dengan budaya, diperlukan, untuk mengecualikan diagnosis bersaing. Kerusakan oleh GVHD diwakili oleh edema, peluruhan mukosa dan pendarahan mungkin, dan histopatologi biasanya mendokumentasikan crypt-sel nekrosis dan putus sekolah dengan abses crypt. Terapi sebagian besar didasarkan pada imunosupresi dan steroid, sehingga risiko infeksi oportunistik harus hati-hati ditimbang, sebagai jumlah kematian akibat komplikasi infeksi telah dilaporkan dalam literatur ( Ferrara et al, 2003. , Jacobsohn & Vogelsang, 2007). Data klinis, epidemiologis dan laboratorium terbatas yang tersedia saat ini mengenai patogenisitas kemungkinan H. alvei. Dalam kasus yang dilaporkan di sini, patogenisitas H. alvei tetap tidak menentu, meskipun temuan dari jumlah bakteri tinggi dan isolasi dari organisme sebagai pertumbuhan murni berat, dan pengobatan terlepas antimikroba dan steroid, perbaikan klinis dan hilangnya organisme dari budaya berjalan pada kecepatan yang sama. Pokoknya, tampaknya menjadi menarik bahwa organisme muncul dalam tinja hanya beberapa minggu

H. Alvei

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sebelum timbulnya gejala, dan menghilang dari feses dalam waktu 10 hari dari resolusi klinis. Peran amikasin dalam pemberantasan kolonisasi tidak jelas, sejak terapi antimikroba tampaknya tidak menjadi sukses sebelum awal anti-GVHD pengobatan. Apakah penggunaan steroid dan senyawa imunosupresif saja, tanpa antimikroba, akan diikuti oleh H. alveipemberantasan dari usus masih belum diketahui. Mungkin, profilaksis kotri memainkan peran luar biasa dalam menggantikan flora normal dengan H. Alvei (perlu dicatat bahwa organisme menunjukkan

resistensi

terhadap

obat

dikutip),

sedangkan

pengobatan

imunosupresif mungkin telah mengubah pertahanan kekebalan mukosa, dengan demikian menyebabkan pertumbuhan berlebih dari organisme. Akhirnya, kerusakan enterik oleh GVHD mungkin telah meningkatkan kepatuhan dari patogen ke epitel, dan pembentukan biofilm, mungkin mengakibatkan kegagalan amikasin untuk memberantas penjajahan sebelum resolusi GVHD. Pada gilirannya, peran H. alvei dalam meningkatkan kerusakan mukosa tidak dapat dikesampingkan. Bahkan, timbulnya infeksi peradangan terkait perubahan mukosa sebagai faktor risiko untuk pengembangan GVHD dapat diduga, seperti crossreaksi

akhirnya

antara

mekanisme

pertahanan

tuan

rumah

(leukosit,

imunoglobulin) dan usus-dinding antigen di satu sisi, dan host respon imun dan H. alvei antigen di sisi lain. Kasus ini semakin menegaskan bahwa perilaku ini sebagai organisme komensal enterik atau patogen yang kontroversial, dan masih banyak pertanyaan yang belum terjawab. Contoh kasus III : Teshima et al. (1992) menemukan penyakit ginjal yang diakibatkan oleh infeksi alami bakteri Hafnia alvei pada juvenil umur setahun ikan cherry salmon Oncorhynchus masou yang dipelihara di kolam ikan lokal di Jepang. Dari luar, ikan yang sakit menunjukkan permukaan tubuh yang gelap dan perut membengkak, dan mereka berenang perlahan-lahan. Dari dalam tubuh, kerusakan jaringan dengan berbagai ukuran, yang tampak seperti benjolan putih keabuan, timbul pada sisi ventral ginjal; secara histopatologis gejala-gejala ini mirip dengan gejala “bacterial kidney disease” (penyakit ginjal bakterial) yang diakibatkat oleh bakteriRenibacterium salmoninarum. Patologi ginjal secara eksperimental bisa

H. Alvei

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ditimbulkan kembali dengan isolat murni Hafnia alvei yang diambil dari luka-luka pada ginjal ikan yang terinfeksi alami. Periode inkubasi penyakit ini pada ikan cherry salmon muda adalah sekitar 3 bulan setelah penyuntikan intraperitoneal tunggal. Penyakit ini, bagaimanapun, bisa muncul lebih cepat sejalan dengan peningkatan frekuensi penyuntikan isolat bakteri.

H. Alvei

Page 14

BAB III PENUTUP

3.1.Kesimpulan Berdasarkan pemaparan Hafnia adalah genus dari Enterobacteriaceae keluarga yang hanya spesies adalah Gram-negatif , fakultatif anaerob , berbentuk batang bakteri Hafnia alvei. Sedangkan Hafnia alvei itu sendiri adalah komensal pada saluran pencernaan manusia dan biasanya tidak patogen , tetapi dapat menyebabkan penyakit pada immunocompromised pasien. Hal ini sering resisten terhadap antibiotik ganda, termasuk aminopenicillins. H. alvei terdapat di berbagai mamalia, ikan, burung, tanah, air, dan sejumlah makanan. H. alvei memiliki mekanisme virulensi beberapa yang berbeda, yang mirip atau identik dengan gram negatif enteropatogen lainnya. Pada manusia, H. alvei merupakan penyebab sejumlah penyakit, termasuk pneumonia, meningitis, abses, dan septicaemia.

3.2. Saran

Hasil yang dikemukakan pada karya tulis ini penulis anggap kurang akurat dikarenakan dalam proses pembuatannya hanya menggunakan referensi dari bacaan dan tidak melalui tahap penelitian. Untuk kedepannya penulis sarankan agar menggunakan metode penelitian. Hal ini akan menambah keakuratan data.

H. Alvei

Page 15

Daftar Pustaka jcm.asm.org/content/37/12/4186.full http://microblog.me.uk/31 http://googel22.blogspot.com/2012/08/cara-membuat-makalah.html http://www.vumicro.com/vumie/help/VUMICRO/Hafnia_alvei.htm http://web2.uwindsor.ca/courses/biology/fackrell/Microbes/5298.htm http://jherrera.sites.truman.edu/hafnia-alvei/ http://menjaga-bumi.blogspot.com/2012/03/makalah-kesehatan.html http://www.retroscope.eu/wordpress/hafnia-alvei/ http://www.uniprot.org/uniprot/P52869

H. Alvei

Page 16

LAMPIRAN

H. Alvei

Page 17

H. Alvei

Page 18

H. Alvei

Page 19

Berikut contoh jurnal yang membahas tentang bakteri Hafnia alvei :

H. Alvei

Page 20

J Lipid Res. 2010 March; 51(3): 564–574. doi: 10.1194/jlr.M001362

PMCID: PMC2817586

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides[S] Jolanta Lukasiewicz,1,* Wojciech Jachymek,* Tomasz Niedziela,* Lennart Kenne,† and Czeslaw Lugowski*§ Author information ► Article notes ► Copyright and License information ►

O-14:0) at O-3′, was identified by

Abstract

ESI-MSn and MALDI-time-of-flight Hafnia

alvei,

bacterium,

a

is

Gram-negative

an

opportunistic

pathogen associated hospital

with

infections,

mixed

bacteremia,

septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity

of

endotoxins

predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first

time.

The

major

form,

asymmetrically hexa-acylated lipid A built of β-d-GlcpN4P-(1→6)-α-dGlcpN1P

substituted

with

(TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of heptaacylated forms of H. alvei lipid A resulted

from

the

addition

of

palmitate (16:0) substituting 14:0(3OH) at N-2 of the α-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.

(R)-

14:0(3-OH) at N-2 and O-3, 14:0(3(R)-O-12:0) at N-2′, and 14:0(3-(R)-

H. Alvei

Page 21

Keywords: Polish Collection of

at O-2 and O-3 of α-d-GlcpN and

Microorganisms, endotoxin, mass

14:0[3-(R)-O-12:0] or 14:0[3-(R)-O-

spectrometry, palmitoylation

14:0] at O-2′ and O-3′ of β-d-GlcpN (2, 3). Such hexa-acylated lipid A

Lipopolysaccharide (LPS, endotoxin) is the main surface antigen (Oantigen) and important virulence factor of most of the Gram-negative bacteria pathogenic for humans and animals (1). LPS contributes greatly to the structural integrity of the bacterial cell wall and constitutes a “pathogen-associated

molecular

pattern” for host infection (1).

are smooth-type molecules built of polysaccharide,

core

oligosaccharide, and lipid A. Among all

these

defined

regions,

the

glycolipid part of LPS called lipid A constitutes a center of biological activity

of

the

endotoxin

that

stimulates different cells of the immune

system

(2).

The

most

common type of lipid A that is also one of the highly endotoxic forms consists of the bisphosphorylated carbohydrate backbone disaccharide β-d-GlcpN4P-(1→6)-α-d-GlcpN1P, substituted by six asymmetrically distributed fatty acids linked via ester and amide linkages: 14:0[3-(R)-OH]

H. Alvei

the

immunostimulatory

highest or

endotoxic

activity in the mammalian host (1, 2) and it was identified for the first time in Escherichia coli LPS (2, 3). Interaction

of

such

asymmetric,

hexa-acylated lipid A region of LPS with mCD14/TLR4/MD-2 receptor complex

on

the

surface

of

monocytes/macrophages constitutes

Most of the known structures of LPS

O-specific

displays

a major mechanism responsible for innate immune response to Gramnegative infection (1, 2). High levels of inflammatory mediators [tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, INFγ, INFα, nitric oxide, platelet-activating factor, and endorphins], as a response of the immune system to large amount of LPS released into a bloodstream, have

profound

cardiovascular

effects system,

on

the

kidneys,

lungs, liver, and central nervous system and trigger the coagulation cascade.

Excessive

inflammatory

response of the innate immune system finally leads to sepsis and septic shock (1).

Page 22

Hafnia

alvei

lipopolysaccharides

cows were reported as a consequence

have been studied for many years

of such infections (4). The clinical

because of frequent reports about

symptoms of H. alvei septicaemia of

opportunistic infections caused by

pullets are similar to those caused by

this bacterium but also several

Salmonella spp. in different host

interesting structural features of their

species (6).

core oligosaccharides and O-specific polysaccharides. As an opportunistic pathogen,

H.

alvei

has

been

associated with respiratory diseases and mixed hospital infections in humans (4). Most cases of H. alveirelated bacteremia usually originate from gastrointestinal and respiratory infections. The bacteremia (mostly monomicrobic infection) and sepsis seem to be the most common syndromes

reported

for

this

bacterium (4). These Gram-negative bacteria were isolated from blood, hepatic

abscesses,

pancreatic

pseudocyst fluid, sputum, feces, and central venous catheter. H. alvei has also been isolated from a variety of foods, e.g., cow milk, honey, corned beef, hard goat cheese, and fish (5). H. alvei infections can cause serious diseases and also substantial loss in farm

animal

production.

Hemorrhagic septicemia in rainbow trout and in laying hens, pneumonic diseases in goats, and mastitis in

Lipopolysaccharides of H. alvei are unusual examples of enterobacterial endotoxins,

among

the

few

elucidated so far, having various Kdo-containing motifs in the outer core

regions

oligosaccharides feature

of

the

core

(7).

Thus,

this

distinguishes

them

from

classical enterobacterial endotoxins. Even

though

many

polysaccharide

O-specific

and

core

oligosaccharide structures have been elucidated for H. alvei LPSs (8–17), the structure of the H. alvei lipid A, the

center

of

toxicity

of

the

endotoxin, has not been analyzed so far. Because the majority of clinical symptoms of diseases caused by this bacterium have LPS-related origin and

the

lipid

A

structure

predominantly affects the biological activity of endotoxins, it seems to be a gap in our knowledge of H. alvei LPSs. Moreover, recent studies on the

invasion

and

intracellular

survival of different H. alvei strains

H. Alvei

Page 23

in a HeLa cell line revealed that

published data (7) connect the lipid

these bacteria were able to enter and

A and core oligosaccharide of H.

persist in human epithelial cells (18).

alvei 32 LPS. Elucidation of the H.

For Gram-negative bacteria that

alvei

employ

survival

precondition for future examination

mechanism during invasion, e.g.,

of a possible relationship between

Salmonella typhimurium (19, 20),

atypical core oligosaccharide (outer

various lipid A modifications are

core Kdo-containing motifs), lipid A

needed.

structure, and biological activity of

intracellular

To

survive

within

mammalian cells, an addition of 4-

lipid

A

structure

is

a

H. alvei LPSs.

amino-4-deoxy-l-arabinose (Ara4N) and phosphoethanolamine (PEtn) and palmitate residues is often required.

MATERIALS AND METHODS Bacteria

Thus, it was interesting to investigate H.

alvei

lipids

A,

the

last

uncharacterized region of their LPS.

H. alvei strains 32, PCM 1192, PCM 1206, and PCM 1207 were obtained from

The

studies

presented

herein

the

Polish

Microorganisms

Collection

(PCM)

at

of the

describe, for the first time, lipid A of

Institute

H. alvei LPS. Recently we have

Experimental

determined the structure of the

Poland). The bacteria were grown in

carbohydrate backbone of H. alvei

Davis medium, killed with 0.5%

32 lipid A as a part of N,O-

phenol, centrifuged using a CEPA

deacylated LPS, which is built of the

flow

bisphosphorylated

suspended in water, and freeze-dried

disaccharide,

GlcpN4P-(1→6)-GlcpN1P (7). We

of

Immunology Therapy

laboratory

and

(Wroclaw,

centrifuge,

(16).

now report on the structural studies of H. alvei 32 and 1192 lipid A

LPS and lipid A isolation and

regions, with emphasis on fatty acid

purification

analysis

and

their

distribution

performed by ESI-MSn and MALDItime-of-flight presented

H. Alvei

(TOF)

results

and

MS.

The

recently

LPS was extracted from bacterial cells by the hot phenol/water method (21). Briefly, freeze-dried bacteria

Page 24

were suspended in 45% phenol

mg/ml) and incubated with stirring at

solution (2g/50 ml) and incubated at

50° C for 5 h followed by drying

65° C with intermittent stirring for

with a stream of nitrogen. The

15 min. The suspension was cooled

partially

down to the temperature below 10° C

samples were further analyzed by

and centrifuged (3,000 g, 30 min).

ESI-MS.

O-deacylated

lipid

A

Water phase was collected. Water was added to compensate for the collected volume of water phase and the cycle was repeated. The water phases were combined and dialyzed against deionized water to remove residual phenol, filtered, and freezedried. The obtained crude LPS was dissolved in water and purified by ultracentrifugation (105,000 g, 6 h) as previously reported (16). Lipid A was isolated from the LPS (200 mg) as a water-insoluble fraction by treatment with 1.5% acetic acid (45 min

at

100°C)

followed

by

centrifugation (40,000 g, 20 min). The

sediment

(lipid

A)

was

resuspended in water and freezedried.

Analytical procedures Prior to analyses, LPS and lipid A were

additionally

extraction

purified

with

by 2:1:3

chloroform/methanol/water mixture (v/v/v)

to

remove

membrane

The

absolute

phospholipids.

configuration of monosaccharides was determined on dephosphorylated and O,N-deacylated lipid A as described by Gerwig et al. (23, 24), using (R)-2-butanol for the formation of

2-butyl

glycosides.

N,O-

deacylation and dephosphorylation of lipid A were performed by hydrolysis with 4 M hydrochloric acid followed by 48% HF treatment as previously described (25). The trimethylsilylated butyl glycosides

Partial O-deacylation of lipid A

were then identified by comparison The partial liberation of the ester-

with

linked fatty acids from lipid A was

carbohydrate standards (Sigma, St.

done by mild alkali treatment (22).

Louis, MO) and (R/S)-2-butanol

Lipid A (0.5 mg) was suspended in

(Fluka, Buchs, Switzerland) on GC-

25%

MS. GC-MS analysis was carried out

H. Alvei

ammonium

hydroxide

(1

samples

produced

from

Page 25

with

a

Hewlett-Packard

5971A

for the analysis of lipid A samples in

system using an HP-1 fused-silica

negative and positive ion modes,

capillary column (0.2 mm × 12 m)

respectively.

and a temperature program from 100

electrospray mass spectra of lipid A

to 270°C at 8°C/min. Qualitative

were recorded using ESQUIRE-LC

analysis of lipid A was done

ion trap mass spectrometer (Bruker

separately for amide- and ester-

Daltonics, Bremen, Germany) for

bound fatty acids using chemical

ESI-MSn analyses of native lipid A

analysis followed by GC-MS (26).

and

Ester-bound fatty acids were released

(Bruker

Daltonics,

Bremen,

from intact LPS by treatment with

Germany)

for

of

sodium CH3ONa in methanol and the

partially deO-acylated lipid A. The

amide-linked

were

lipid A samples were desalted and

released from deO-acylated LPS by

dissolved in a methanol/chloroform

aqueous 4 M KOH at 120°C for 16 h.

mixture (1:1, v/v, 1 mg/ml). In ESI-

The absolute configuration of 3-

IT MSn analyses, the samples were

hydroxy fatty acids was determined

continuously infused through the

by GC-MS (HP-5 column, 0.25 mm

capillary head at 4 kV into the ion

× 30 m; temperature program from

source using a linear syringe pump at

150 to 270°C at 8°C/min) using 3-

a rate of 2 μl/min. Spectra were

methoxy-(S)-phenylethylamide

scanned in the m/z 200–2200 range.

derivatives (27).

The mass isolation window for the

fatty

acids

Negative-ion

micrOTOF-Q

spectrometer

analysis

the

precursor ion selection was set to 4 Mass spectrometry MALDI-TOF MS was carried out on a Kratos Kompact-SEQ instrument as described previously (25). 9HPyrido[3,4-b]indole [10 mg/ml in a 1:1 acetonitrile/water mixture (v/v)] and

2,4,6-trihydroxyacetophenone

[25 mg/ml in a 1:1 acetonitrile/water mixture (v/v)] were used as matrices

H. Alvei

Da in all the MSn analyses. In analyses performed on micrOTOF-Q spectrometer,

the

samples

dissolved

in

a

were 1:1

methanol/chloroform mixture (v/v, 1 mg/ml) and analyzed by direct infusion at a rate of 3 μl/min with an electrospray capillary high voltage of 4,5 kV. Spectra were scanned in the

Page 26

ranges of m/z 100–2250 (MS) and

dried.

The

yield

of

lipid

A

m/z 250–2000 (MS2 of an ion at m/z

preparations were between 11% and

1291.9). The ion source temperature

16% of LPS.

was 200°C, the N2 flow rate was set at 4 l/min, and the pressure of nitrogen was

0.4 bar. External

calibration in the negative-ion mode was

applied

Tunemix™(neg)

using mixture

the (Bruker

Daltonics, Germany) in quadratic regression mode and m/z range of 113–2234 Da. The isolation width for MS experiments was Δm/z = 10,

Compositional analysis Chemical analyses of lipid A from H. alvei 32 and PCM 1192 indicated the presence of GlcN, phosphate group (P group), dodecanoic acid (12:0), tetradecanoic acid (14:0), 3-hydroxytetradecanoic acid [14:0(3-OH)] and hexadecanoic acid (16:0) in the

2

and the collision energy of the quadrupole was −45 eV.

relative

proportions

2.0:1.6:0.9:1.0:3.7:0.1.

GC-MS

analysis of the trimethylsilylated (R)2-butyl

RESULTS

glycosides

of

sugar

constituents of lipid A showed the Isolation of lipopolysaccharide and

presence of the d-GlcN isomer.

lipid A

Qualitative analysis of fatty acids

LPS of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 were extracted from bacterial cells by the hot phenol/water

method

(21)

and

purified as previously described (16). The yields of LPS preparations were 2%–3.5%. Lipid A was liberated by acidic hydrolysis of LPS (1.5% acetic acid, 45 min, at 100°C). The reaction mixture was cooled and centrifuged.

The

sediment

was

resuspended in water and freeze-

H. Alvei

was also done separately for amideand ester-linked fatty acids. GC-MS data revealed the presence of (R)14:0(3-OH), 14:0, 12:0, and 16:0 as ester-bound fatty acids and (R)14:0(3-OH) as the only amide-linked fatty acid. Identification of the methoxy derivative, the methyl ester of

3-methoxytetradecanoic

acid,

among fatty acid methyl esters obtained

by transestrification

of

acyloxyacyl residues in lipid A with sodium methanolate, indicated that

Page 27

some of 14:0(3-OH) fatty acids were

seventh acyl residue. The lipid A

substituted with the “secondary”

variant represented by ions at m/z

fatty acid in the native lipid A.

1768.3 explained

ESI-MS/MSn

analyses of lipid A

ESI mass spectra obtained for lipid A isolated from H. alvei 32, 1192, 1206,

and

1207

showed

their

heterogeneity and identical pattern of ions (Fig. 1), thus suggesting a common structure of the lipid A. On the basis of compositional analyses and taking into account previously published structure of the N,Odeacylated H. alvei 32 LPS (7), the most abundant ions at m/z 1796.4 and m/z 1716.5 could be attributed to bis- and mono-phosphorylated hexaacylated forms of lipid A carrying four (R)-14:0(3-OH), one 14:0 and one 12:0 fatty acids (Fig. 1A). Additional forms of lipid A were also identified but observed ions were of much lower intensity. Ions at m/z 1954.6 and 2034.6 could be attributed

to

mono-

and

bis-

phosphorylated hepta-acylated forms of lipid A, respectively (Fig. 1, inset

and by

1688.4 bis-

phosphorylated

could and

be

mono-

hexa-acylated

molecules that are substituted by one shorter fatty acid in comparison with the major form described above (mass difference of 28 Da observed between ions at m/z 1796.4 and 1768.3 and at m/z 1716.5 and 1688.4). The peaks at lower m/zvalues and intensities could represent different bisphosphorylated lipid A species devoid of 14:0 (m/z 1586.1), 14:0 and 14:0(3-OH) (m/z 1360.1) or monophosphorylated species devoid of 14:0 (m/z 1506.3), 14:0(3-OH) (m/z 1490.2), and 14:0 and 14:0(3OH) (m/z 1279.9) (Fig. 1). The presence of ions with lower m/z values

and

those

related

to

monophosphorylated forms can be explained by partial degradation of lipid A during work-up and lability of some acyl side chains and the P group at O-1 during the acid hydrolysis (28).

structure). The mass difference 238 Da between m/z 1954.6 and m/z 1716.5 indicates the presence of hexadecanoic acid (16:0) as the

H. Alvei

Page 28

of studied lipid A, ESI-MSn spectra were compared with data published previously for lipid A of E. coli (29– 31)

.Fig. 2.

Fig. 1. Negative ion mode ESI mass spectra obtained for lipid A (inset structures) isolated from LPSs of H. alvei 1192 (A), 32 (B), 1206 (C), and 1207 (D). The ions denoted with an asterisk correspond to a mass difference of 28 due to the decrease in carbon

Negative ion mode ESI-MS2 of lipid A from H. alvei 32 (A) and 1192 (B) represented by the ion at m/z 1716.5 (outlined Interpretation

inset of

structure). the

observed

fragment ions is presented in the inset structures. The representation

chain ...

0,4

A2 is according ...

The distribution of fatty acids was determined for the lipid A isolated from H. alvei 32 and PCM 1192 by multiple-stage ESI-MSn (n = 2 and 3). Detected ions were interpreted according to the rules described previously in ESI-MSn studies of lipid A (25, 29). ESI-MS2 was performed on the selected ions at m/z 1716.5 (Fig. 2), m/z 1506.3 (Fig. 3A) and m/z 1279.9 (supplementary Fig. I).

Because

the

obtained

data

Fig. 3. Negative ion mode ESI-MSn of lipid A from H. alvei 32 represented by an

suggested the E. coli-type structure

H. Alvei

Page 29

the ion at m/z 1506.3 (outlined inset

14:0) as a ketene derivative], m/z

structure). A: MS2 of the ion at m/z

1261.8 [elimination of 14:0(3-O-

1506.3. B: MS3 of the ion at m/z

14:0) as fatty acid]. The most

1261.8 (form I and II). Interpretation

abundant daughter ion at m/z 1243.8,

of

corresponded to the elimination of

observed

fragment

ions

is

presented ...

two ester-linked fatty acids, 14:0(3OH) at O-3 and secondary-bound

To determine the structure of the most

abundant

component

corresponding to the most abundant ion (m/z 1716.5) in lipid A from both H. alvei 32 and PCM 1192, this ion

14:0 at O-3′ (Fig. 2, outlined inset structure). Two double bonds were formed

as

a

result

of

the

eliminations. The mass of the ion at m/z 1243.8 was in agreement with

1

was isolated in MS and fragmented in MS2 (Fig. 2, outlined inset structure). The spectra of lipid A from both strains showed almost identical pattern of daughter ions. Chemical

analysis

of

lipid

A

constituents suggested that this ion could

be

attributed

to

the

monophosphorylated, hexa-acylated lipid A form containing two GlcN, one P group, four 14:0(3-OH), one 14:0, and one 12:0 (Fig. 2, outlined inset structure). Ions at m/z 1516.1, m/z

1488.0,

and

m/z

1472.0,

correspond to elimination of 12:0, 14:0 and 14:0(3-OH), respectively, from the parent ion. Product ions from elimination of two acyl groups were also detected: m/z 1287.8 (elimination of 12:0 and 14:0), m/z

the calculated mass of two GlcN (one unsaturated), one P group, two 14:0(3-OH) fatty acids, one 12:0 fatty acid, and one 14:1 Δ2 fatty acid derivative. Subsequent loss of a 14:1 Δ2 from O-3′ as a ketene derivative and

free

fatty

acid

yielded

a

characteristic pair of ions at m/z 1035.8 and m/z 1017.7, respectively (Fig. 2). The observed elimination of 14:1 Δ2 by both charge-driven (loss of a ketene derivative) and chargeremote processes (loss of a free fatty acid) suggested its location at O-3′ and also its previous substitution by 14:0 (ion at m/z 1243.8). The ions at m/z 708.4 and 690.4 represented 0,4

A2 fragments [according to the

nomenclature

of

Domon

and

Costello (32)] and could be formed

1279.9 [elimination of 14:0(3-O-

H. Alvei

Page 30

from the ions at m/z 1035.8 and

outlined inset structure). Almost

1017.7, respectively. These ions

identical spectra were obtained for

corresponded to the in-source intra-

the lipid A of H. alvei 1192 (data not

ring fragmentation and confirmed the

shown). Peaks with lower intensity at

distribution of the identified fatty

m/z

acids. Interpretation of these types of

correspond to elimination of water

ions makes it possible to define fatty

and 12:0 fatty acid, respectively. The

acid distribution on each GlcN

most abundant ion at m/z 1261.8

residue. In the case of MS2 of the ion

could be the result of elimination of

at m/z 1716.5, such fragments were

14:0(3-OH) at O-3 or O-3′ as a free

identified only for daughter ions and

fatty acid. Because elimination of the

not for the isolated parent ion. Thus,

acyloxyacyl group at O-3′ of the

an

distal GlcN could occur by both

interpretation

of

intra-ring

1488.3

and

m/z

1305.9

fragmentation ions was described

charge-driven

(elimination

as

a

below for MSn analyses of ions with

ketene derivative) and charge-remote

lower m/z ratio (MS2 of the ions at

processes (elimination as free fatty

m/z 1506.3 and 1279.9, and MS3 of

acid), the pair of ions at m/z 1279.8

the ion at m/z 1261.8).

and m/z 1261.8 corresponds to such elimination of 14:0(3-OH) at O-3′.

Further investigation of fatty acid distribution was performed by MS2 and MS3 of the ion at m/z 1506.3 observed in MS analysis of lipid A preparations from both H. alvei 32 and PCM 1192 (Fig. 3A). On the basis of chemical analysis and types of

the

observed

fragment

ions

(described below), the ion at m/z 1506.3 could represent pentaacylated lipid A that is monophosphorylated

Elimination of both 14:0(3-OH) from O-3 as a free fatty acid and from O3′ as a free fatty acid and a ketene derivative

corresponds

to

the

characteristic pair of ions at m/z 1035.8 and 1017.7. The presence of 0,2

A2 (m/z 1221.0) and

2,5

A2 (m/z

1202.6) fragments of the parent ion confirmed the substitution of GlcN by 14:0(3-OH) fatty acid at N-2 (Fig. 3A, inset structures).

at O-4′, consisting of two GlcN, one P group, four 14:0(3-OH) fatty acids,

The most abundant ion at m/z 1261.8

and one 12:0 fatty acid (Fig. 3A,

derived from the MS2 fragmentation

H. Alvei

Page 31

was selected for MS3 in order to

m/z

determine the distribution of the

elimination of 14:0(3-OH) as free

remaining fatty acids (Fig. 3B). The

fatty acid at O-3 (form II), which

m/z value of this isolated ion was in

explains its higher abundance in

agreement with the calculated mass

comparison with the ion at m/z

of two GlcN, one P group, three

1035.8. Ions of lower intensity

14:0(3-OH), and one 12:0 fatty acid.

correspond to molecules that have

Considering the high stability of acyl

lost 14:0(3-OH), have a double bond

amides, the ion at m/z 1261.8 was

at O-3, and a hydroxyl group or

attributed to two forms of the

double bond at O-3′. Several types of

following

diagnostic fragment ions, e.g.

structure:

monophosphorylated

I) lipid

a A,

1017.7

(m/z 976.9),

could

0,4

arise

from

A2 (m/z 690.4),

0,2

A2

2,5

A2

substituted with 14:0(3-OH) at N-2

(m/z 958.8), C1 (m/z 648.0), and Y1

and O-3′ and 14:0(3-O-12:0) at N-2′

(m/z 630.0), were detected from the

and II) a monophosphorylated lipid

parent ion (Fig. 3B, outlined inset

A, substituted with 14:0(3-OH) at N-

structures). These ions confirmed the

2 and O-3 and 14:0(3-O-12:0) at N-

location of the 14:0(3-OH) at N-2,

2′ (Fig. 3B, outlined inset structures).

O-3 or O-3′ and 14:0(3-O-12:0) at N-

These two possible variants differ by

2′.

the presence or lack of 14:0(3-OH) at

elimination of 12:0 from amide-

O-3 or O-3′. Obtained MS3 spectrum

bound 14:0(3-O-12:0) were also

contained ions originating from both

detected (m/z 835.5 from the ion at

forms. The peak at m/z 1243.8 is

m/z 1035.8 and m/z 817.5 from the

consistent with elimination of water

ion at m/z 1017.7), but due to the

(−18 Da). Subsequent elimination of

higher stability of acyl amides, the

14:0(3-OH) at O-3′ (form I) yielded

peaks from these ions exhibited very

a characteristic pair of ions at m/z

low intensities. Two structural forms

1035.8 and 1017.7, corresponding to

for the parent ion (m/z 1261.8) and

the loss of a 14:0(3-OH) as a ketene

interpretation of ions corresponding

derivative (−180 Da) or the loss of a

to the in-source intra- and inter-ring

free

Da),

fragmentation are presented in Fig.

respectively. Alternatively, the ion at

3B. The observed pattern of ions

H. Alvei

fatty

acid

(−198

Ions

corresponding

to

the

Page 32

confirmed that lipid A, represented

of

by the fragment ion at m/z 1506.3, is

presented ...

a

monophosphorylated

disaccharide,

observed

fragment

ions

is

GlcN

substituted

with

14:0(3-OH) at N-2, O-3 and O-3′ and

To determine the structure of the most

abundant

component

corresponding to the most abundant

14:0(3-O-12:0) at N-2′.

ion (m/z 1716.5) in lipid A from both ESI-MSn analysis (supplemental Fig.

H. alvei 32 and PCM 1192, this ion

I) of the fatty acid distribution in

was isolated in MS1 and fragmented

lipid A from both strains was also

in MS2 (Fig. 2, outlined inset

performed for the most abundant ion,

structure). The spectra of lipid A

m/z 1279.9, in the low mass region of

from both strains showed almost

the ESI mass spectrum (Fig. 1).

identical pattern of daughter ions.

Observed fragment ions indicate that

Chemical

this

constituents suggested that this ion

ion

could

tetraacylated

lipid

represent A.

a

ESI-MS2

could

analysis

be

of

attributed

lipid

to

A

the

analysis indicated that this lipid A

monophosphorylated, hexa-acylated

form of H. alvei, represented by the

lipid A form containing two GlcN,

ion

the

one P group, four 14:0(3-OH), one

structure,

14:0, and one 12:0 (Fig. 2, outlined

and

inset structure). Ions at m/z 1516.1,

at

m/z

glucosamine

1279.9, backbone

phosphorylated

at

has

O-4′

substituted with 14:0(3-OH) at N-2

m/z

1488.0,

and

m/z

1472.0,

and O-3, and 14:0(3-O-12:0) at N-2′

correspond to elimination of 12:0,

(supplementary Fig. I, outlined inset

14:0 and 14:0(3-OH), respectively,

structure).

from the parent ion. Product ions from elimination of two acyl groups

Fig. 3.

were also detected: m/z 1287.8 n

Negative ion mode ESI-MS of lipid A from H. alvei 32 represented by an the ion at m/z 1506.3 (outlined inset structure). A: MS2 of the ion at m/z

(elimination of 12:0 and 14:0), m/z 1279.9 [elimination of 14:0(3-O14:0) as a ketene derivative], m/z 1261.8 [elimination of 14:0(3-O-

3

1506.3. B: MS of the ion at m/z 1261.8 (form I and II). Interpretation

H. Alvei

14:0) as fatty acid]. The most abundant daughter ion at m/z 1243.8,

Page 33

corresponded to the elimination of

distribution of the identified fatty

two ester-linked fatty acids, 14:0(3-

acids. Interpretation of these types of

OH) at O-3 and secondary-bound

ions makes it possible to define fatty

14:0 at O-3′ (Fig. 2, outlined inset

acid distribution on each GlcN

structure). Two double bonds were

residue. In the case of MS2 of the ion

formed

the

at m/z 1716.5, such fragments were

eliminations. The mass of the ion at

identified only for daughter ions and

m/z 1243.8 was in agreement with

not for the isolated parent ion. Thus,

the calculated mass of two GlcN

an

(one unsaturated), one P group, two

fragmentation ions was described

14:0(3-OH) fatty acids, one 12:0

below for MSn analyses of ions with

fatty acid, and one 14:1 Δ2 fatty acid

lower m/z ratio (MS2 of the ions at

derivative. Subsequent loss of a 14:1

m/z 1506.3 and 1279.9, and MS3 of

Δ2 from O-3′ as a ketene derivative

the ion at m/z 1261.8).

and

free

as

a

result

fatty

acid

of

yielded

1035.8 and m/z 1017.7, respectively (Fig. 2). The observed elimination of 14:1 Δ2 by both charge-driven (loss of a ketene derivative) and chargeremote processes (loss of a free fatty acid) suggested its location at O-3′ and also its previous substitution by 14:0 (ion at m/z 1243.8). The ions at m/z 708.4 and 690.4 represented A2 fragments [according to the

nomenclature

of

Domon

of

intra-ring

a

characteristic pair of ions at m/z

0,4

interpretation

and

Costello (32)] and could be formed from the ions at m/z 1035.8 and 1017.7, respectively. These ions corresponded to the in-source intraring fragmentation and confirmed the

Further investigation of fatty acid distribution was performed by MS2 and MS3 of the ion at m/z 1506.3 observed in MS analysis of lipid A preparations from both H. alvei 32 and PCM 1192 (Fig. 3A). On the basis of chemical analysis and types of

the

observed

fragment

ions

(described below), the ion at m/z 1506.3 could represent pentaacylated lipid A that is monophosphorylated at O-4′, consisting of two GlcN, one P group, four 14:0(3-OH) fatty acids, and one 12:0 fatty acid (Fig. 3A, outlined inset structure). Almost identical spectra were obtained for the lipid A of H. alvei 1192 (data not shown). Peaks with lower intensity at

H. Alvei

Page 34

m/z

1488.3

and

m/z

1305.9

agreement with the calculated mass

correspond to elimination of water

of two GlcN, one P group, three

and 12:0 fatty acid, respectively. The

14:0(3-OH), and one 12:0 fatty acid.

most abundant ion at m/z 1261.8

Considering the high stability of acyl

could be the result of elimination of

amides, the ion at m/z 1261.8 was

14:0(3-OH) at O-3 or O-3′ as a free

attributed to two forms of the

fatty acid. Because elimination of the

following

acyloxyacyl group at O-3′ of the

monophosphorylated

distal GlcN could occur by both

substituted with 14:0(3-OH) at N-2

charge-driven

a

and O-3′ and 14:0(3-O-12:0) at N-2′

ketene derivative) and charge-remote

and II) a monophosphorylated lipid

processes (elimination as free fatty

A, substituted with 14:0(3-OH) at N-

acid), the pair of ions at m/z 1279.8

2 and O-3 and 14:0(3-O-12:0) at N-

and m/z 1261.8 corresponds to such

2′ (Fig. 3B, outlined inset structures).

elimination of 14:0(3-OH) at O-3′.

These two possible variants differ by

Elimination of both 14:0(3-OH) from

the presence or lack of 14:0(3-OH) at

O-3 as a free fatty acid and from O-

O-3 or O-3′. Obtained MS3 spectrum

3′ as a free fatty acid and a ketene

contained ions originating from both

derivative

the

forms. The peak at m/z 1243.8 is

characteristic pair of ions at m/z

consistent with elimination of water

1035.8 and 1017.7. The presence of

(−18 Da). Subsequent elimination of

0,2

A2 (m/z

14:0(3-OH) at O-3′ (form I) yielded

1202.6) fragments of the parent ion

a characteristic pair of ions at m/z

confirmed the substitution of GlcN

1035.8 and 1017.7, corresponding to

by 14:0(3-OH) fatty acid at N-2 (Fig.

the loss of a 14:0(3-OH) as a ketene

3A, inset structures).

derivative (−180 Da) or the loss of a

(elimination

corresponds

A2 (m/z 1221.0) and

as

to

2,5

free The most abundant ion at m/z 1261.8

fatty

structure:

acid

I) lipid

(−198

a A,

Da),

respectively. Alternatively, the ion at

2

derived from the MS fragmentation

m/z

1017.7

could

arise

from

3

was selected for MS in order to determine the distribution of the remaining fatty acids (Fig. 3B). The

elimination of 14:0(3-OH) as free fatty acid at O-3 (form II), which explains its higher abundance in

m/z value of this isolated ion was in

H. Alvei

Page 35

comparison with the ion at m/z

14:0(3-OH) at N-2, O-3 and O-3′ and

1035.8. Ions of lower intensity

14:0(3-O-12:0) at N-2′.

correspond to molecules that have lost 14:0(3-OH), have a double bond at O-3, and a hydroxyl group or double bond at O-3′. Several types of diagnostic fragment ions, e.g. 0,4

(m/z 976.9),

A2 (m/z 690.4),

0,2

A2

2,5

A2

(m/z 958.8), C1 (m/z 648.0), and Y1 (m/z 630.0), were detected from the parent ion (Fig. 3B, outlined inset structures). These ions confirmed the location of the 14:0(3-OH) at N-2,

ESI-MSn analysis (supplemental Fig. I) of the fatty acid distribution in lipid A from both strains was also performed for the most abundant ion, m/z 1279.9, in the low mass region of the ESI mass spectrum (Fig. 1). Observed fragment ions indicate that this

ion

could

tetraacylated

lipid

represent A.

a

ESI-MS2

analysis indicated that this lipid A

O-3 or O-3′ and 14:0(3-O-12:0) at N-

form of H. alvei, represented by the

2′.

ion

Ions

corresponding

to

the

elimination of 12:0 from amidebound 14:0(3-O-12:0) were also detected (m/z 835.5 from the ion at m/z 1035.8 and m/z 817.5 from the ion at m/z 1017.7), but due to the higher stability of acyl amides, the peaks from these ions exhibited very low intensities. Two structural forms for the parent ion (m/z 1261.8) and interpretation of ions corresponding to the in-source intra- and inter-ring fragmentation are presented in Fig. 3B. The observed pattern of ions confirmed that lipid A, represented by the fragment ion at m/z 1506.3, is a

monophosphorylated

disaccharide,

H. Alvei

substituted

GlcN with

at

m/z

glucosamine

1279.9,

has

backbone

phosphorylated

at

the

structure, O-4′

and

substituted with 14:0(3-OH) at N-2 and O-3, and 14:0(3-O-12:0) at N-2′ (supplementary Fig. I, outlined inset structure). To confirm that the identified fatty acid

distribution

in

monophosphorylated form of H. alvei lipid A follows the same pattern as bisphosphorylated and hexa-acylated molecule, the ion at m/z 1797.3 was isolated and ESIMS2 analysis was carried out for H. alvei 32 (Fig. 4B) and H. alvei PCM 1192 (supplementary Fig. II). Thus, the

results

confirmed

identical

Page 36

distribution of fatty acids in the bisphosphorylated forms of lipid A isolated

from

both

strains

(supplementary Fig. II). Product ions were formed mainly by the loss of phosphoric acid at O-1 (m/z 1698.3) with subsequent elimination of esterbound acyl and acyloxyacyl groups [14:0 (m/z 1470.1), 14:0(3-OH) (m/z 1454.1),

14:0(3-O-14:0)

(m/z

1243.9)] (Fig. 4B). The frequency of the loss of phosphoric acid from the

Fig. 4.

position O-1 was significantly higher

Negative ion mode ESI-MS2 of lipid

than from O-4′ during ESI-MS2

A isolated from H. alvei 32 LPS. A:

analysis due to the lability of

The MS2 of the ion at m/z 2034.6

glycosidically linked P group (28,

(bisphosphorylated

31). Interpretation of the observed

acylated lipid A, outlined inset

fragment ions is presented in Fig. 4

structure). B: The MS2 spectrum of

(inset structures). Ion at m/z 1498.1

the

attributed to the elimination of

(bisphosphorylated

secondary-bound 12:0 fatty acid at

acylated ...

N-2′

exhibited

low

ion

at

and

m/z and

hepta-

1797.3 hexa-

intensity.

Elimination of acyl residues with

The results of compositional analysis

preservation of the P group at O-1

of H. alvei 32 lipid A and the mass

was also observed (m/z 1568.0,

difference (238 Da) between the ion

1552.1, 1323.8).

at m/z 1797.3 (bisphosphorylated and hexa-acylated lipid A) and an ion at m/z 2034.6 (bisphosphorylated and hepta- acylated lipid A) suggested the presence of a 16:0 fatty acid in hepta-acylated lipid A. To confirm that 16:0 is a secondary-linked fatty

H. Alvei

Page 37

acid

substituting

amide-linked

14:0(3-OH) at N-2, MS2 analysis of

ESI-MS analysis of partially deOacylated lipid A

the ion at m/z 2034.6 was performed (Fig. 4A). Observed fragmentation was similar to that observed for the ion

at

m/z

1797.3

(Fig.

4B).

Fragment ions were formed mainly by the loss of the glycosidically linked P group and elimination of primary and secondary, ester-bound acyl and acyloxyacyl groups at O-3 and O-3′ [14:0 (m/z 1708.5), 14:0(3OH) (m/z 1692.4), 14:0(3-O-14:0) (m/z 1500.1/1482.2)]. Elimination of acyl residues with the preservation of P group at O-1 was also observed (m/z 1806.5, 1790.4, 1580.3, 1562.2, 1336.2). Several ions resulting from elimination of 16:0 fatty acid were identified (e.g., m/z 1778.4, 1680.4, 1436.2, 1208.0), further supported the presence of 16:0 in the analyzed structure. The mass difference (200 Da) between pairs of related ions m/z 999.7/981.7 and m/z 799.6/781.7 indicated that 12:0 is a substituent of amide-linked 14:0(3-OH) at N-2′ of the

bisphosphorylated,

acylated lipid A.

hepta-

ESI-MS and ESI-MS2 analyses of partially

deO-acylated

lipid

A

samples allowed the determination of type and location of the seventh fatty acid in the hepta-acylated forms of H. alvei lipid A. The NH4OH treatment leads to partial liberation of ester-linked acyl and acyloxyacyl residues (22, 33). Negative-ion mass spectra of the partially deO-acylated lipid A (Fig. 5A) followed by MS2 analysis (Fig. 5B) gave further information

concerning

hepta-

acylated forms of lipid A from H. alvei 32 and 1192. Several molecular ion peaks were observed at m/z 1371.9, m/z 1291.9, m/z 1133.7, m/z 1053.7, and m/z 951.5 together with their doubly charged variants (Fig. 5A, inset structures). Two of them could

be

explained

by

the

bisphosphorylated (m/z 1371.9) and monophosphorylated (m/z 1291.9) tetra-acylated lipid A that were formed by hydrolysis of 14:0(3-OH) and 14:0(3-O-14:0) at O-3 and O-3′, respectively, from the hepta-acylated lipid A. The ion at m/z 1263.9, contributing

to

an

additional

microheterogeneity of the lipid A

H. Alvei

Page 38

(mass difference of 28 Da). The MS

mono-

and

bis-phosphorylated

analysis also revealed the presence of

molecules. Arrows ...

ion at m/z 453.4 that corresponded to the

deprotonated

14:0(3-O-14:0)

released upon NH4OH hydrolysis.

To confirm the presence of 16:0 as a secondary fatty acid at N-2 of the proximal GlcN, MS2 analysis of an ion at m/z 1291.9 was performed with the use of an ESI-(Q)-TOF mass spectrometer (Fig. 5B). The fragmentation pattern observed for the singly charged ion at m/z 1291.9 revealed the structure showed in Fig. 5B (inset structure), corresponding to the

GlcN

disaccharide

monophosphorylated at O-4′ and substituted with 14:0(3-O-16:0) and 14:0(3-O-12:0) at N-2 and N-2′, respectively. Two ions at m/z 1035.7 and m/z 1091.7 could correspond to elimination

of

16:0

and

12:0,

respectively. Subsequent loss of both fatty acids yielded the ion at m/z 835.5 (Fig. 5B, inset structure). The diagnostic fragment ions, e.g., Fig. 5. A: Negative ion mode ESI-MS mass spectrum obtained for partially deOacylated lipid A (inset structures) isolated from LPS of H. alvei 32. The difference of 80 Da corresponds to the difference in mass between

(m/z 768.4),

0,4

A2 (m/z 708.4),

0,2

A2

2,5

A2

(m/z 750.4), C1 (m/z 666.4), and B1 (m/z 648.4) supported the presence of amide-bound 14:0(3-O-12:0) at N2′ and the free hydroxyl group at position O-3. Most of these ions had analogs resulting from elimination of 12:0

(m/z

568.3,

508.2,

466.2,

448.2). Several ions corresponded to

H. Alvei

Page 39

molecules devoid of one or two

abundant

ions

represented

secondary 12:0 and/or 16:0 fatty

sodium [M+Na]+ (m/z 1741) and

acids (m/z 1053.7, 871.5, 853.4) and

potassium

this could be a result of dissociation

adducts of the monophosphorylated

of solvated deprotonated species at

form of hexa-acylated lipid A and

the skimmer of electrospray source

the B1+ ions (m/z 1087 and 887),

(31). The presence of described ions

arising from the cleavage of the

combined with the ESI-MSn analysis

glycosidic linkage between the two

of native lipid A allowed the

GlcN residues at high laser power

determination of the seventh fatty

settings. The ion at m/z 1087

acid as a constituent of the acyl

corresponds to the average mass of

substituent 14:0(3-O-16:0) at N-2.

the

[M+K]+

phosphorylated

(m/z

the

1757)

tetraacylated

GlcN, substituted with 14:0(3-OMALDI-TOF analysis

12:0) at N-2′ and 14:0(3-O-14:0) at

The structural information provided by the negative ESI-MS analysis was compared with MALDI-TOF MS data

to

confirm

the

proposed

structure of lipid A and above all, the substitution positions of 14:0(3-OH)

O-3′. The B1+ ion devoid of the secondary-bound 12:0 fatty acid at N-2′ was also detected (m/z 887). Elimination of the secondary fatty acid at N-2′ is preferred in positiveion mode MS in comparison to

at O-3 and 14:0 at O-3′ as the

negative-ion mode experiments (34).

“secondary” ester-linked fatty acids.

The presence of such ions provided

These fatty acids were eliminated first

during

experiments.

the

ESI-MS2

MALDI-TOF

mass

spectra of H. alvei 32 lipid A obtained at high laser power in the positive-ion

mode

showed

additional evidence that the N-2′ amide-bound 14:0(3-OH) fatty acid is substituted by a 12:0 and not by a 14:0 fatty acid. DISCUSSION

the

presence of the oxonium ion B1+,

The lipopolysaccharide constitutes

which identified the fatty acids that

the “pathogen-associated molecular

substitute the β-d-GlcN4P residue

pattern” for host infection by Gram-

(supplementary Fig. III). The most

negative bacteria and is among the

H. Alvei

Page 40

most powerful natural activators of

atoms (Fig. 1), conforming to a

the innate immune system (1). Lipid

classical form of enterobacterial-type

A, the center of biological activity of

lipid A. The analyzed lipids A of H.

the

last

alvei are identical with those of E.

uncharacterized region of endotoxins

coli (2, 3), S. typhimurium (35, 36),

isolated from H. alvei.

Serratia

LPS,

has

been

the

marcescens

(37),

Providencia rettgeri (37), Klebsiella To complete the recently published structure of the N,O-deacylated LPS of H. alvei 32 (7), the lipid A isolated

from

LPS

32

was

investigated by qualitative analysis of fatty acids and their location in the lipid A disaccharide backbone was established. Comparative analysis performed by MS suggested that LPS of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 have the same

oxytoca (33), and Shigella flexneri Sc576 (msbB mutant) (29). The carbohydrate backbone and primarylinked fatty acids of H. alvei lipid A are also similar to those of Klebsiella pneumoniae,

Proteus

Aeromonas

sp.,

mirabilis, Haemophilus

influenzae, Campylobacter jejuni, Shigella

sonnei,

Yersinia

pestis,

Enterobacter agglomerans, Ervinia corotovora, and Rhizobium etli (36).

lipid A structure (Fig. 1, inset structure).

The described form of lipid A warrants

We have now demonstrated that lipid A of H. alvei LPS consists of hexaand

hepta-acylated

molecules.

Detailed mass spectrometric analysis carried out on lipid A isolated from LPS of H. alvei 32 and PCM 1192 showed that their major form was built

of

β-GlcpN4P-(1→6)-α-

maximal

immunostimulatory

activities

of

LPSs (2). Because most of H. alvei LPSs studied to date are smooth-type molecules and have E. coli-type lipid A structure, they would most likely induce high inflammatory response of the innate immunity system during sepsis as well.

GlcpN1P substituted with saturated “primary” (R)-3-hydroxylated and

The

“secondary” nonhydroxylated acyl

forms of H. alvei lipid A resulted

residues of 12 and mostly 14 carbon

from the addition of a palmitate

H. Alvei

presence

of

hepta-acylated

Page 41

(16:0) to 14:0(3-OH) at N-2 of

Salmonella, E. coli, L. pneumophila,

GlcpN. This type of a single

B. bronchiseptica, Y. enterocolitica,

modification of lipid A, called

and Y. pseudotuberculosis (41). The

palmitoylation,

palmitoylation, together with the

observed

was

only

previously

in

a

few

regulated addition of Ara4N and

enterobacterial lipids A of S. enterica

PEtn,

serovar Typhimurium (38), E. coli

environmental conditions and can

(39), and K. pneumoniae (40).

directly protect the bacterium from

Similar modification of an acyl

certain host immune defenses (41).

moiety of lipid A was also observed

Such a modification attenuates the

in

ability of lipid A to activate defense

Legionella

pneumophila,

is

dependent

mechanisms

bronchiseptica,

Yersinia

signal transduction pathway (41) and

Yersinia

provides

and

the

the

Pseudomonas aeruginosa, Bordetella

enterocolitica,

through

on

resistance

TLR4

to

certain

pseudotuberculosis LPS, but in most

cationic antimicrobial peptides (38).

of the cases the palmitoylation

The

position was different (41). It is

presumably

known that palmitoylated lipid A

hydrophobic and van der Waals

usually

interactions in the outer membrane of

coexists

as

a

addition

palmitate

increases

the

other lipid A variants. A palmitate

translocation

chain

a

antimicrobial peptides across the

phospholipid is incorporated into

bilayer. The palmitoylation helps

lipid A by an outer membrane

bacteria to maintain and monitor the

enzyme (PagP). The PagP enzyme

outer membrane permeability and

transfers palimitate chain from the

lipid

sn-1 position of a phospholipid to the

Salmonella, as an enteropathogen,

hydroxyl group of the 14:0(3-OH) at

utilizes lipid A modifications to

N-2 of proximal GlcpN of lipid A

survive within macrophages (41).

(41).

Studies of the immune signaling in

The

gene

pagP

from

and

its

wall, of

asymmetry

functional homologs were identified

human

among several bacteria, for example

chemically

H. Alvei

thus,

the

substoichiometric component with

originating

cell

of

cell

lines

preventing

the

as

cationic

well

showed

synthesized

(41).

that hepta-

Page 42

acylated lipid A has 10- to 100-fold

(18). Thus, H. alvei lipid A structure

lower activity in comparison with its

elucidation and the identification of

hexa-acylated analogs (42).

hepta-acylated variant among other forms of H. alvei lipid A are

Some LPSs of H. alvei and K. pneumoniae

share

their

general

important for the understanding of the pathogenicity of this bacterium.

structure, having structural Kdocontaining motifs in the outer core

Acknowledgments

region (7) and a basic lipid A structure. LPS isolated from the polymyxin-resistant K. pneumoniae O3 mutant contained approximately five times more of the 4-amino-4deoxy-L-arabinopyranose

and

an

The

authors

thank

Marek

Jon,

Faculty of Chemistry, University of Wroclaw, for his help and assistance with the ESI-Q-TOF measurements. Footnotes

increased ratio of hepta-acylated lipid A (the addition of 16:0 fatty

Abbreviations:

acid) compared with that of a

LPS

polymyxin-sensitive

parent

strain

lipopolysaccharide

P group

phosphate group

(40). PCM Polish Collection Inasmuch as many of the already known bacteria

cases

of

of

Microorganisms

Gram-negative

employing

the

TOF

time-of-flight

palmitoylation of lipid A regulate

This work was supported by grants

this process through the PhoP/PhoQ-

No. 6 PO 04A 069 19 from the State

activated pagP gene or its homologs

Committee of Scientific Research

(41), it is possible that H. alvei could

(KBN), Poland, and the Swedish

regulate this process in a similar

Research Council. The collaboration

way. Recent studies showed that

between Polish and Swedish groups

some H. alvei strains were able to

was supported by funds from The

enter and persist in human epithelial

Royal Swedish Academy of Sciences

cells, but little is known about factors

and

contributing to this invasion strategy

Sciences. Part of this work was

H. Alvei

The

Polish

Academy

of

Page 43

presented at the 3rd German-Polish-

4. Janda J. M., Abbott S. L. 2006.

Russian

Bacterial

The genus Hafnia: from soup to nuts.

Poland,

Clin. Microbiol. Rev. 19: 12–18.

Meeting

Carbohydrates,

on

Wroclaw,

October 6–9, 2004.

[PMC free article] [PubMed] 5. Casagrande Proietti P., Passamonti

[S]

The online version of this article

(available

at

http://www.jlr.org)

contains supplementary data in the form of three figures.

F., Pia Franciosini M., Asdrubali G. 2004. Hafnia alvei infection in pullets in Italy. Avian Pathol. 33: 200–204. [PubMed] 6. Kelly W. R. 1993. Patterns of

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