RESUMO
The peritrophic matrix (PM) is an acellular membrane that covers the gut epithelium in arthropods and physically separates it from the lumen. The structure is thought to play an important role in tick biology. The PM is also known to impact the persistence of tick-borne pathogens like Borrelia burgdorferi, although limited information is available about its molecular constituents or their biological significance. Herein, we characterise a novel PM-associated gut protein in Ixodes scapularis ticks, annotated as Peritrophic Membrane Chitin Binding Protein (PM_CBP), for its role in the integrity and function of the matrix. The PM_CBP displays homology to the chitin deacetylase metalloenzyme, shows upregulation during tick feeding, and is localized at the luminal surface of the gut epithelium. The structural integrity of the PM was impaired both by the knock down of PM_CBP expression via RNA interference and by treatment with anti-PM_CBP antibodies, as revealed by its electron microscopic appearance. Additionally, the duration of tick engorgement on mice and the passage of experimentally-inoculated fluorescent dextran molecules across the PM are affected by the knock down of PM_CBP expression. The transfer of anti-PM_CBP antibodies into the tick gut impacted the overall composition of the resident microbiome, and also influenced B. burgdorferi acquisition in ticks and its transmission to mice. Taken together, these data highlight the biological significance of the Ixodes PM and suggest that the targeting of its molecular constituents may contribute to the development of novel interventions against tick-borne infections.
Assuntos
Proteínas de Artrópodes/metabolismo , Borrelia burgdorferi/fisiologia , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Ixodes/metabolismo , Ixodes/microbiologia , Doença de Lyme/microbiologia , Animais , Borrelia burgdorferi/patogenicidade , Proteínas de Transporte/metabolismo , Quitina/metabolismo , DNA Bacteriano , Feminino , Técnicas de Silenciamento de Genes , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Ligação Proteica , Interferência de RNA , RNA Ribossômico 16SRESUMO
Borrelia burgdorferi is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of B. burgdorferi, orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.
Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/imunologia , Evasão da Resposta Imune , Lipoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Animais , Antígenos de Bactérias/imunologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Vetores Aracnídeos/microbiologia , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Células Cultivadas , Proteínas do Sistema Complemento/imunologia , Citocinas/biossíntese , Citocinas/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Ixodes/microbiologia , Lipoproteínas/genética , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos SCID , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface-exposed, conserved, and potential multi-domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host-pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region-specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N-terminal and middle regions and a combined middle and C-terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain-specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N-terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Processamento de Proteína Pós-Traducional , Serina Proteases/metabolismo , Fatores de Virulência/metabolismo , Animais , Camundongos , Proteólise , CarrapatosRESUMO
In China, Leptospira interrogans serovar Lai strain 56601 (str.56601) is one of main pathogenic strains that cause severe leptospirosis in both human and animals. The genome of this organism was completely sequenced in 2003. However, in 2011, we identified and corrected some assembly errors in the str.56601 genome due to the repeat sequences widely distributed in the Leptospira genome. In this study, we re-analyzed the previously reported mobile, phage-related genomic island in the chromosome and rectified detailed sequence information in both the plasmid and chromosome using various experimental methods. The presence of a separate circular extrachromosomal plasmid was also confirmed, and its location in the genomic region was determined relative to the genomic island reported in L. interrogans serovar Lai by a combination of pulsed-field gel electrophoresis -based and plasmid extraction-based Southern blot analysis. This report confirmed that the separate extrachromosomal circular plasmid is not integrated into the chromosome of L. interrogans str.56601 and markedly improved our understanding of the genomic organization, evolution, and pathogenesis of L. interrogans. In particular, characterization of this extrachromosomal circular plasmid will contribute to the development of genetic manipulation systems in pathogenic Leptospira species.
Assuntos
Cromossomos Bacterianos , Leptospira interrogans/genética , Plasmídeos , Sequência de Bases , Primers do DNA , Leptospira interrogans/patogenicidadeRESUMO
Brain organoids with nucleus-specific identities provide unique platforms for studying human brain development and diseases at a finer resolution. Despite its essential role in vital body functions, the medulla of the hindbrain has seen a lack of in vitro models, let alone models resembling specific medullary nuclei, including the crucial spinal trigeminal nucleus (SpV) that relays peripheral sensory signals to the thalamus. Here, we report a method to differentiate human pluripotent stem cells into region-specific brain organoids resembling the dorsal domain of the medullary hindbrain. Importantly, organoids specifically recapitulated the development of the SpV derived from the dorsal medulla. We also developed an organoid system to create the trigeminothalamic projections between the SpV and the thalamus by fusing these organoids, namely human medullary SpV-like organoids (hmSpVOs), with organoids representing the thalamus (hThOs). Our study provides a platform for understanding SpV development, nucleus-based circuit organization, and related disorders in the human brain.
Assuntos
Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Borrelia burgdorferi/crescimento & desenvolvimento , Citratos/farmacologia , Doença de Lyme/diagnóstico , Animais , Anticoagulantes/farmacologia , Borrelia burgdorferi/efeitos dos fármacos , Meios de Cultura/química , Modelos Animais de Doenças , Humanos , Doença de Lyme/microbiologia , Camundongos , Camundongos SCID , Sensibilidade e Especificidade , Citrato de SódioRESUMO
Acute liver failure (ALF) is a severe clinical syndrome characterized by massive death of hepatocytes in a short time, resulting in coagulopathy and hepatic encephalopathy, with a high mortality in patients without pre-existing liver disease. Effective treatment of ALF is currently limited to liver transplantation, highlighting the need for new target therapies. Here, we found that expression of hepatic tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor tumor necrosis factor receptor superfamily member 12A (Tnfrsf12a) were significantly increased during ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Inhibition of TWEAK/Tnfrsf12a axis markedly attenuated TAA or APAP-induced ALF. Moreover, our results demonstrated that TWEAK/Tnfrsf12a axis induced receptor-interacting protein kinase 1 (RIPK1)-dependent apoptosis of hepatocytes, instead of necroptosis or pyroptosis. Notably, hepatic TNFRSF12A and TWEAK levels were also significantly increased in liver biopsies from ALF patients. In summary, our results demonstrate that during ALF, TWEAK/Tnfrsf12a axis activates RIPK1 in hepatocytes, leading to RIPK1-dependent apoptosis and subsequent liver injury. Therefore, inhibition of either TWEAK/Tnfrsf12a axis or RIPK1-dependent apoptosis attenuates liver injury, providing a new potential therapeutic target for the treatment of ALF.
RESUMO
Protein kinase-mediated phosphorylation plays a critical role in many biological processes. However, the identification of key regulatory kinases is still a great challenge. Here, we develop a trans-omics-based method, central kinase inference, to predict potentially key kinases by integrating quantitative transcriptomic and phosphoproteomic data. Using known kinases associated with anti-cancer drug resistance, the accuracy of our method denoted by the area under the curve is 5.2% to 29.5% higher than Kinase-Substrate Enrichment Analysis. We further use this method to analyze trans-omic data in hepatocyte maturation and hepatic reprogramming of human dermal fibroblasts, uncovering 5 kinases as regulators in the two processes. Further experiments reveal that a serine/threonine kinase, PIM1, promotes hepatic conversion and protects human dermal fibroblasts from reprogramming-induced ferroptosis and cell cycle arrest. This study not only reveals new regulatory kinases, but also provides a helpful method that might be extended to predict central kinases involved in other biological processes.
Assuntos
Ferroptose , Ciclo Celular , Pontos de Checagem do Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos , Ferroptose/genética , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismoRESUMO
High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5'-monophosphate and 3'-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5' snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.
RESUMO
BACKGROUND: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV). RESULTS: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites. CONCLUSIONS: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.
Assuntos
Vetores Aracnídeos/genética , Borrelia burgdorferi/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Ixodes/genética , Transportadores de Ânions Orgânicos/genética , Animais , Vetores Aracnídeos/microbiologia , Vetores Aracnídeos/virologia , Borrelia burgdorferi/patogenicidade , Linhagem Celular , Biologia Computacional , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Expressão Gênica , Ixodes/química , Ixodes/microbiologia , Ixodes/virologia , Ninfa/microbiologia , Ninfa/virologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sulfimpirazona/farmacologia , Transaminases/genética , Viroses , Xanturenatos/metabolismoRESUMO
The proteome of Borrelia burgdorferi undergoes dynamic alterations as the microbe cycles through and persists in diverse host or vector environments. Therefore, studies of B. burgdorferi proteome and protein-protein interactions, which play central roles in biological processes in diverse organisms, are critical in understanding biology and infectivity of spirochetes. Here, we describe the proteomic analysis of B. burgdorferi by two-dimensional (2-D) gel electrophoresis followed by protein identification via liquid chromatography-mass spectrometry and database searching. We also describe assays for studying the interaction between borrelial proteins: a novel high-throughput luciferase assay, yeast two-hybrid assay, and a far-Western assay that are routinely used in our laboratories.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Doença de Lyme/microbiologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas de Bactérias/análise , Far-Western Blotting/métodos , Borrelia burgdorferi/química , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Proteoma/análise , Proteoma/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The global increase in multidrug resistant (MDR) bacteria has led to phage therapy being refocused upon. A novel endolysin, LysPA26, containing a lysozyme-like domain, was screened against Pseudomonas aeruginosa in this study. It had activity against MDR P. aeruginosa without pretreatment with an outer-membrane permeabilizer. LysPA26 could kill up to 4 log units P. aeruginosa in 30 min. In addition, temperature and pH effect assays revealed that LysPA26 had good stability over a broad range of pH and temperatures. Moreover, LysPA26 could kill other Gram-negative bacteria, such as Klebsiella pneumonia, Acinetobacter baumannii and Escherichia coli, but not Gram-positive bacteria. Furthermore, LysPA26 could eliminate P. aeruginosa in biofilm formation. Our current results show that LysPA26 is a new and promising antimicrobial agent for the combat of Gram-negative pathogens.
RESUMO
Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp.
Assuntos
Adaptação Fisiológica/fisiologia , Genoma Bacteriano/fisiologia , Leptospira/genética , Fatores de Virulência/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leptospirose/genéticaRESUMO
Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.
Assuntos
Leptospira/genética , Plasmídeos/análise , Southern Blotting , China , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Leptospira/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Pathogenic Leptospira spp. causes leptospirosis in China and throughout the world. Here, we have sequenced two L. interrogans moderately virulent vaccine strains JDL03 (serovar Canicola) and JDL10 (serovar Hebdomadis) used in China. We selected a subproteomic approach to identify surface-exposed proteins including OMPs and extracellular proteins of these two strains plus a highly virulent vaccine strain 56601 (serovar Lai). Comparative surface-exposed proteome among the three strains indicated 81 cores, 61 dispensable and 122 unique surface-exposed proteins. Finally, the 10 highly conserved surface-exposed or subsurface proteins included two known cross-reactive antigens (LipL32 and LA_3469) and another two novel antigens (LA_0136 and LA_0505) displaying conserved immunoreactivity among 15 Chinese epidemic serovars. Furthermore, many potential virulence factors were detected in these identified surface-exposed proteins, such as Loa22, LipL32, LenC, LenF and OmpL37. Interestingly, LipL45, ClpA and ClpB, exhibiting obvious amino acid mutations among str.56601, str.JDL03 and JDL10, might contribute to virulence differences observed among these strains. Additionally, specific surface-exposed proteins in virulent str.56601 were considered to be key virulence determinants, such as Zn-dependent protease, cholesterol oxidase precursor, and so on. In all, we had relatively complete surface-exposed subproteomes of L. interrogans, which will enhance our understanding of leptospiral pathogenesis and key virulence determinants. BIOLOGICAL SIGNIFICANCE: The present work demonstrates the use of genomic sequencing and subproteomic studies for the identification of potential vaccine and diagnostic antigen candidates against leptospirosis. The data show the conserved surface-exposed proteins to be novel potentially vaccine/diagnostic candidates. Furthermore, the data also show that LipL45, ClpA, ClpB and a lipoprotein from these three strains plus another highly virulent strain Fiocruz L1-130 contain specific amino acid mutations in strains JDL03 and JDL10. The surface-exposed subproteome of pathogenic L. interrogans could provide valuable information to gain a more complete understanding of leptospiral pathogenesis and virulence determinants.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/metabolismo , Leptospira interrogans/metabolismo , Leptospira interrogans/patogenicidade , Proteoma/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Reações Cruzadas , Leptospira interrogans/genética , Leptospira interrogans/imunologia , Leptospirose/genética , Leptospirose/imunologia , Leptospirose/metabolismo , Leptospirose/patologia , Proteoma/genética , Proteoma/imunologia , Proteômica , Coelhos , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
BACKGROUND: Previous genomic analysis of pathogenic Leptospira has identified two circular chromosomes but no plasmid. This study aims to investigate potential extrachromosomal elements of L.interrogans serovar Canicola strain Gui44. METHODOLOGY: Two novel plasmids, pGui1 and pGui2, were isolated from the pathogenic strain Gui44, using a modified alkaline lysis method. Southern blotting was performed to determine the presence and size of them. Then, 454 and Hiseq sequencing were applied to obtain and analyze the complete sequences of the two plasmids. Furthermore, real-time quantitative PCR and next-generation sequencing were used to compare relative copy numbers of the two plasmids with that of the chromosomes. Finally, after serial passages in vitro for more than 2 years, the strain Gui44 was subsequently re-sequenced to estimate stability of the two plasmids. PRINCIPAL FINDINGS: The larger plasmid, pGui1, 74,981 base pairs (bp) in length with GC content of 34.63%, possesses 62 open reading frames (ORFs). The smaller plasmid, pGui2, is 66,851 bp in length with GC content of 33.33%, and contains 63 ORFs. The replication initiation proteins encoded by pGui1 and pGui2 demonstrate significant sequence similarity with LA1839 (86% and 88%), a well-known replication protein in another pathogenic L.interrogans serovar Lai strain Lai, suggesting the ability for autonomous plasmid replication. Quantitative PCR and next-generation sequencing confirms a single copy of both plasmids and their stable presence in the strain Gui44 with in vitro serial passages after more than 2 years. Interestingly, the two plasmids both contain a significant number of novel genes (35 in pGui1 and 52 in pGui2). CONCLUSIONS: This report confirms the presence of two separate circular plasmids in serovar Canicola strain Gui44 and provides a new understanding of genomic organization, adaptation, evolution and pathogenesis of Leptospira, which will aid in the development of in vivo genetic manipulation systems in pathogenic Leptospira species.
Assuntos
Genoma Bacteriano/genética , Leptospira interrogans serovar canicola/genética , Plasmídeos/genética , Dados de Sequência Molecular , SorogrupoRESUMO
Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.