Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans.
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ABSTRACT:
SUBMITTER: Santos JC
PROVIDER: S-EPMC7744396 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors.
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Project description:Fifty-three strains belonging to the pathogenic species Leptospira interrogans and Leptospira kirschneri were analyzed by multilocus sequence analysis. The species formed two distinct branches. In the L. interrogans branch, the phylogenetic tree clustered the strains into three subgroups. Genogroups and serogroups were superimposed but not strictly.
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Project description:Leptospirosis is a worldwide zoonotic infection of human and veterinary concern. Caused by pathogenic spirochetes of the genus Leptospira, the disease presents greater incidence in tropical and subtropical regions. The identification of proteins that could be involved in the bacteria host interactions may facilitate the search for immune protective antigens. We report the proteomic analysis of Leptospira interrogans serovar Pomona virulent strain LPF cultured from kidney and liver of infected hamsters. Total protein extracts were separated by two-dimensional gel electrophoresis (2-DE), 895 spots were analyzed by MALDI-TOF mass spectrometry (MS), and 286 were identified as leptospiral proteins, corresponding to 108 distinct proteins. These proteins are allocated in all the bacterial cell compartments and are distributed in every functional category. Furthermore, the previously described, known outer membrane proteins, OmpL1, LipL21, LipL31, LipL32/Hap-1, LipL41, LipL45, LipL46, LruA/LipL71, and OmpA-like protein Loa22 were all recognized. Most importantly, this research work identified 27 novel leptospiral proteins annotated as hypothetical open reading frames (ORFs). We report for the first time an array of proteins of the Leptospira expressed by virulent, low-passage strain. We believe that our studies, together with the genome data will enlighten our understanding of the disease.
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Project description: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.
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Project description:Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. Using two independent screening methods, mass spectrometry and protein arrays, members of the cadherin family were identified as potential L. interrogans receptors on mammalian host surfaces. We focused our investigation on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VE-cadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans.
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Project description:A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown to specifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activity was encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could be identified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. The apparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas open reading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processing at the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and protein levels with Staphylococcus aureus beta-hemolysin and sphingomyelinase C from three Bacillus cereus strains was observed. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.
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Project description:The Leptospira interrogans ponA and pbpB genes were isolated and characterized. ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins from Escherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA and pbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized in E. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.
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Project description:Leptospira, the causative agent of leptospirosis is known to have several proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. In this study, we carried out in-depth proteomic analysis of Triton X-114 fractions of Leptospira interrogans using high-resolution LC-MS/MS.
2022-02-17 | PXD026044 | Pride
Project description:The first step of lipid A biosynthesis is catalyzed by LpxA in Escherichia coli (EcLpxA), an acyltransferase selective for UDP-GlcNAc and R-3-hydroxymyristoyl-acyl carrier protein (ACP). Leptospira interrogans LpxA (LiLpxA) is extremely selective for R-3-hydroxylauroyl-ACP and an analogue of UDP-GlcNAc, designated UDP-GlcNAc3N, in which NH(2) replaces the GlcNAc 3-OH group. EcLpxA does not discriminate between UDP-GlcNAc and UDP-GlcNAc3N; however, E. coli does not make UDP-GlcNAc3N. With LiLpxA, R-3-hydroxylauroyl-methylphosphopantetheine efficiently substitutes for R-3-hydroxylauroyl-ACP. We now present crystal structures of free LiLpxA and its complexes with its product UDP-3-N-(R-3-hydroxylauroyl)-GlcNAc3N and with its substrate R-3-hydroxylauroyl-methylphosphopantetheine. The positions of the acyl chains of the R-3-hydroxylauroyl-methylphosphopantetheine and the UDP-3-N-(R-3-hydroxylauroyl)-GlcNAc3N are almost identical and are similar to that of the acyl chain in the EcLpxA/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc complex. The selectivity of LiLpxA for UDP-GlcNAc3N may be explained by the orientation of the backbone carbonyl group of Q68, which differs by approximately 82 degrees from the corresponding Q73 carbonyl group in EcLpxA. This arrangement provides an extra hydrogen-bond acceptor for the 3-NH(2) group of UDP-GlcNAc3N in LiLpxA. The R-3-hydroxylauroyl selectivity of LiLpxA is explained by the position of the K171 side chain, which limits the length of the acyl-chain-binding groove. Our results support the role of LiLpxA H120 (which corresponds to EcLpxA H125) as the catalytic base and provide the first structural information about the orientation of the phosphopantetheine moiety during LpxA catalysis.
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Project description:We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.
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