Complete type III secretion system of a mesophilic Aeromonas hydrophila strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91

Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.     

Melanization and pathogenicity in the insect, Tenebrio molitor, and the crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.     

Complete Type III Secretion System of a Mesophilic Aeromonas hydrophila Strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

Definition of the extracellular proteome of pathogenic-phase Histoplasma capsulatum

The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease. Within the mammalian host, pathogenic Histoplasma yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to Histoplasma virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC-MS/MS analysis of the proteins in pathogenic-phase culture filtrates of Histoplasma. In addition to secreted Cbp1, the extracellular proteome of pathogenic Histoplasma yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp's). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic Histoplasma mycelia, suggesting that these proteins are linked to Histoplasma pathogenesis. In addition, Histoplasma yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how Histoplasma alters the host-pathogen interaction to its advantage.     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.     

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD , was identified by its ability to complement the pilD mutation in P. aeruginosa . Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes ( tapABCD ) that are homologous to P. aeruginosa type IV pilus biogenesis genes ( pilABCD ). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N -methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila     

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.     

YplA is exported by the Ysc, Ysa, and flagellar type III secretion systems of Yersinia enterocolitica

Yersinia enterocolitica maintains three different pathways for type III protein secretion. Each pathway requires the activity of a specific multicomponent apparatus or type III secretion system (TTSS). Two of the TTSSs are categorized as contact-dependent systems which have been shown in a number of different symbiotic and pathogenic bacteria to influence interactions with host organisms by targeting effector proteins into the cytosol of eukaryotic cells. The third TTSS is required for the assembly of flagella and the secretion of the phospholipase YplA, which has been implicated in Y. enterocolitica virulence. In this study, YplA was expressed from a constitutive promoter in strains that contained only a single TTSS. It was determined that each of the three TTSSs is individually sufficient for YplA secretion. Environmental factors such as temperature, calcium availability, and sodium chloride concentration affected the contribution of each system to extracellular protein secretion and, under some conditions, more than one TTSS appeared to operate simultaneously. This suggests that some proteins might normally be exported by more than one TTSS in Y. enterocolitca.     

Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that injects virulence effector proteins into host cells via a type III secretion system (TTSS). TTSS-deficient mutants have a Hrp- phenotype, that is, they cannot elicit the hypersensitive response (HR) in non-host plants or pathogenesis in host plants. Mutations in effector genes typically have weak virulence phenotypes (apparently due to redundancy), but deletion of six open reading frames (ORF) in the DC3000 conserved effector locus (CEL) reduces parasitic growth and abolishes disease symptoms without affecting function of the TTSS. The inability of the DeltaCEL mutant to cause disease symptoms in tomato was restored by a clone expressing two of the six ORF that had been deleted: CEL ORF3 (HopPtoM) and ORF4 (ShcM). A DeltahopPtoM::nptII mutant was constructed and found to grow like the wild type in tomato but to be strongly reduced in its production of necrotic lesion symptoms. HopPtoM expression in DC3000 was activated by the HrpL alternative sigma factor, and the protein was secreted by the Hrp TTSS in culture and translocated into Arabidopsis cells by the Hrp TTSS during infection. Secretion and translocation were dependent on ShcM, which was neither secreted nor translocated but, like typical TTSS chaperones, could be shown to interact with HopPtoM, its cognate effector, in yeast two-hybrid experiments. Thus, HopPtoM is a type III effector that, among known plant pathogen effectors, is unusual in making a major contribution to the elicitation of lesion symptoms but not growth in host tomato leaves.     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.     

Virulence of Erwinia amylovora,a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses

Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram‐negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D‐DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level.     

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.