Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus

Abstract Ovine footrot is a debilitating and highly infectious disease that is primarily caused by the Gram-negative, anaerobic bacterium Dichelobacter nodosus . The major antigens implicated in virulence are the type IV fimbriae and extracellular proteases. The fimbriae show sequence and structural similarity to other type IV fimbriae, this similarity extends to genes that are involved in fimbrial biogenesis. Several acidic and basic extracellular serine proteases are produced by both virulent and benign isolates of D. nodosus . Subtle functional differences in these proteases appear to be important in virulence. In addition, there are two chromosomal regions that have a genotypic association with virulence. The partially duplicated and rearranged vap regions appear to have arisen from the insertion of a plasmid into a tRNA gene via an integrase-mediated site-specific insertion event. The 27 kb vrl region has several genes often found on bacteriophages and has inserted into an ssrA gene that may have a regulatory role in the cell. The determination of the precise role that each of these genes and gene regions has in virulence awaits the development of methods for the genetic analysis and manipulation of D. nodosus .     

The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.     

The Interaction of Protein Structure, Selection, and Recombination on the Evolution of the Type-1 Fimbrial Major Subunit (fimA) from Escherichia coli

Fimbrial adhesins allow bacteria to interact with and attach to their environment. The bacteria possibly benefit from these interactions, but all external structures including adhesins also allow bacteria to be identified by other organisms. Thus adhesion molecules might be under multiple forms of selection including selection to constrain functional interactions or evolve novel epitopes to avoid recognition. We address these issues by studying genetic diversity in the Escherichia coli type-1 fimbrial major subunit, fimA. Overall, sequence diversity in fimA is high (π= 0.07) relative to that in other E. coli genes. High diversity is a function of positive diversifying selection, as detected by d _N/d _S ratios higher than 1.0, and amino acid residuces subject to diversifying selection are nonrandomly clustered on the exterior surface of the peptide. In addition, McDonald and Kreitman tests suggest that there has been historical but not current directional selection at fimA between E. coli and Salmonella. Finally, some regions of the fimA peptide appear to be under strong structural constraint within E. coli, particularly the interior regions of the molecule that is involved in subunit to subunit interaction. Recombination also plays a major role contributing to E. coli fimA allelic variation and estimates of recombination (2N _e c) and mutation (2N _eμ) are about the same. Recombination may act to separate the diverse evolutionary forces in different regions of the fimA peptide. Received: 13 April 2000 / Accepted: 28 October 2000     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

HapX-Mediated Iron Homeostasis Is Essential for Rhizosphere Competence and Virulence of the Soilborne Pathogen Fusarium oxysporum

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.     

A Putative Transmembrane Leucine Zipper of Agrobacterium VirB10 Is Essential for T-Pilus Biogenesis but Not Type IV Secretion

The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system is composed of a translocation channel and an extracellular T pilus. Bitopic VirB10, the VirB7 lipoprotein, and VirB9 interact to form a cell envelope-spanning structural scaffold termed the “core complex” that is required for the assembly of both structures. The related pKM101-encoded core complex is composed of 14 copies each of these VirB homologs, and the transmembrane (TM) α helices of VirB10-like TraF form a 55-Å-diameter ring at the inner membrane. Here, we report that the VirB10 TM helix possesses two types of putative dimerization motifs, a GxxxA (GA₄) motif and two leucine (Leu1, Leu2) zippers. Mutations in the Leu1 motif disrupted T-pilus biogenesis, but these or other mutations in the GA₄ or Leu2 motif did not abolish substrate transfer. Replacement of the VirB10 TM domain with a nondimerizing poly-Leu/Ala TM domain sequence also blocked pilus production but not substrate transfer or formation of immunoprecipitable complexes with the core subunits VirB7 and VirB9 and the substrate receptor VirD4. The VirB10 TM helix formed weak homodimers in Escherichia coli, as determined with the TOXCAT assay, whereas replacement of the VirB10 TM helix with the strongly dimerizing TM helix from glycophorin A blocked T-pilus biogenesis in A. tumefaciens. Our findings support a model in which VirB10''s TM helix contributes to the assembly or activity of the translocation channel as a weakly self-interacting membrane anchor but establishes a heteromeric TM-TM helix interaction via its Leu1 motif that is critical for T-pilus biogenesis.     

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.     

Spink13, an Epididymis-specific Gene of the Kazal-type Serine Protease Inhibitor (SPINK) Family,Is Essential for the Acrosomal Integrity and Male Fertility

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.     

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein.Helicobacter pylori infects 50% of the world population. Stomach infection with this bacterium is associated with the development of several gastric diseases, including chronic active gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. Factors influencing disease outcomes are not completely understood, but bacterial, host, and environmental factors have been identified that affect the dynamics of this bacterium-host interaction (30). A hallmark of H. pylori infection is the induction of mucosal inflammation, which is a risk factor for developing more severe pathology (27).Epidemiological studies have established that infection with strains harboring the cag pathogenicity island (PAI) leads to a higher risk for development of severe disease (27). The cag PAI size varies between 35 and 40 kb and encodes 27 putative proteins (1, 13). Several of the encoded proteins share sequence similarities with components of the prototypical type IV secretion (T4S) system VirB/D4 of Agrobacterium tumefaciens (15, 16). Based on research done in A. tumefaciens, the components of the molecular machinery have been divided into channel or core complex components (VirB6, VirB7, VirB8, VirB9, and VirB10), energetic components (VirB11, VirB4, and VirD4), and extracellular appendage components (VirB2 and VirB5). VirB6, VirB8, and VirB10 are components anchored at the inner membrane with domains spanning the periplasm, while VirB7 and VirB9 are located at the outer membrane. Energetic components are located at the inner membrane, and pilus components include the main subunit VirB2 and accessory components, such as VirB5, which functions as an adhesin (15, 16). The VirB/D4 T4S is thought to be energized by the inner membrane ATPases, and this energy is transduced to VirB10 and the outer membrane complex for protein translocation (11). The lipoprotein VirB7 is critical for the stability of HpVirB9 at the outer membrane (19).While the extent of homology of the H. pylori cag T4S components is often limited, sequence analysis has allowed the identification of the VirB11 (HP0525 and HpVirB11), VirB10 (HP0527 and HpVirB10), VirB9 (HP0528 and HpVirB9), and VirD4 (HP0524 and HpVirD4) homologues as summarized in Table S1 of the supplemental material (1, 13, 28). HpVirB9 and HpVirB10 homologies are not distributed along the entire length of the protein. For example, HpVirB10 is a very large protein with only a short domain similar to VirB10. HpVirB10 is also reported to localize on the external surface of the pilus (31), while VirB10 is tethered in the inner membrane. HP0529 (HpVirB6) and HP0530 (HpVirB8) have been assigned as homologs of VirB6 and VirB8, respectively (28). HP0523 (HpVirB1) has lytic transglycosylase activity, supporting its designation as a VirB1 homolog (38). HP0532 (HpVirB7) has a lipoprotein attachment site, suggesting a role as a VirB7 homolog (1, 28), and has been suggested to stabilize a Cag T4S outer membrane subcomplex containing CagM, HpVirB9, and HpVirB10 (28).The activity of the cag PAI-encoded T4S system is responsible for the translocation of the effector protein CagA and induction of proinflammatory chemokine and cytokine secretion, including the chemokine interleukin-8 (IL-8) (7). CagA T4S-mediated translocation into host cells is followed by tyrosine phosphorylation on specific tyrosine phosphorylation motifs (EPIYA motifs) at the C-terminal region of the protein and both phosphorylation-dependent and -independent interference with host cellular pathways. The induction of proinflammatory chemokine production is mediated by a still-uncharacterized Cag T4S-mediated delivery of peptidoglycan into host cells and subsequent activation of Nod receptors (37), and it has also been reported that CagA itself has proinflammatory properties (9). The molecular mechanisms responsible for Cag T4S system assembly and activity remain unclear.Null alleles of the genes with homology to T4S components (HpVirB11, HpVirB4, HpVirB6, HpVirB7, HpVirB8, HpVirB9, and HpVirB10) abolish both CagA translocation and IL-8 induction, with the exception of HpVirD4, which affects CagA translocation but not IL-8 induction (20). Other genes of the island also essential for Cag T4S function do not share sequence or structural homology with known T4S components. More detailed analysis of these Cag T4S essential genes allowed the recent assignment of several proteins as functional homologs of additional VirB components. HP0546 was suggested as a VirB2 homolog, the main subunit of other T4S system pili (3). Ultrastructural work suggested that HpVirB10 is also a major subunit of the Cag T4S system pilus (31, 35), but clear evidence that either HpVirB2 or HpVirB10 is the main pilus subunit is still lacking. CagL (HP0539) has been identified (29) as an adhesin (functionally similar to VirB5) whose binding to host cell receptors is required for activation of the secretion process, and CagF (HP0543) has been characterized as a CagA chaperone (17). CagD (HP0545) has been recently reported as a multifunctional Cag T4S component essential for CagA translocation and full IL-8 secretion induction (12).We have characterized the biochemical role of an additional essential H. pylori-specific gene, HP0522/cag3, in Cag T4S. A previous yeast two-hybrid screen that investigated interactions among cag PAI proteins suggested Cag3 could interact with HpVirB8, HpVirB7, CagM (HP0537), and CagG (HP0542) (10). To begin to understand the molecular basis of Cag3 function in T4S we investigated the subcellular localization of the Cag3 protein and the protein-protein interactions this protein establishes in H. pylori cells. We found evidence suggesting that Cag3 is an integral part of the Cag T4S outer membrane subcomplex required to maintain HpVirB7 levels.     

The Response Regulator FimR Is Essential for Fimbrial Production of the Oral Anaerobe Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative oral anaerobe considered to be a major pathogen in adult periodontitis. One of the noted virulence factors of this bacterium is its unique fimbriae, which are composed of FimA (fimbrilin) as a major subunit. We have recently identified and isolated two essential genes, fimS and fimR, for fimbriation of P. gingivalis from transposon-mutagenesis studies. The genes encode two components of a His–Asp phosphorelay system, FimS as a sensor histidine kinase and FimR as a response regulator. Disruption of either gene causes fimbrial deficiency in this organism. In this study, the expression of FimR protein was detected in various P. gingivalis strains. In addition, a fragment containing fimR with a possible promoter was introduced into the fimR -disruption mutant, using a shuttle vector, pT-COW. The transconjugant recovered both FimR and FimA expression at levels comparable to the parentP. gingivalis ATCC 33277. Furthermore, characteristic fimbrial structures were clearly observed around the cell surface of both parent and transconjugant cells under electron microscopy. This is the first successful complementation experiment in P. gingivalis. These results show that the FimR protein is essential as a positive regulator in fimbriation of P. gingivalis.     

Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).     

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.     

A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.     

The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.