Mark P. Stevens

Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways

We have determined the complete genome sequences of a host-promiscuous Salmonella enterica serovar Enteritidis PT4 isolate P125109 and a chicken-restricted Salmonella enterica serovar Gallinarum isolate 287/91. Genome comparisons between these and other Salmonella isolates indicate that S. Gallinarum 287/91 is a recently evolved descendent of S. Enteritidis. Significantly, the genome of S. Gallinarum has undergone extensive degradation through deletion and pseudogene formation. Comparison of the pseudogenes in S. Gallinarum with those identified previously in other host-adapted bacteria reveals the loss of many common functional traits and provides insights into possible mechanisms of host and tissue adaptation. We propose that experimental analysis in chickens and mice of S. Enteritidis-harboring mutations in functional homologs of the pseudogenes present in S. Gallinarum could provide an experimentally tractable route toward unraveling the genetic basis of host adaptation in S. enterica.

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature‐tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI‐4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI‐4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many ‘housekeeping’ genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.

An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell‐to‐cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi‐Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi‐Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa‐secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage‐like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.

Microglial brain region−dependent diversity and selective regional sensitivities to aging

Microglia have critical roles in neural development, homeostasis and neuroinflammation and are increasingly implicated in age-related neurological dysfunction. Neurodegeneration often occurs in disease-specific, spatially restricted patterns, the origins of which are unknown. We performed to our knowledge the first genome-wide analysis of microglia from discrete brain regions across the adult lifespan of the mouse, and found that microglia have distinct region-dependent transcriptional identities and age in a regionally variable manner. In the young adult brain, differences in bioenergetic and immunoregulatory pathways were the major sources of heterogeneity and suggested that cerebellar and hippocampal microglia exist in a more immune-vigilant state. Immune function correlated with regional transcriptional patterns. Augmentation of the distinct cerebellar immunophenotype and a contrasting loss in distinction of the hippocampal phenotype among forebrain regions were key features during aging. Microglial diversity may enable regionally localized homeostatic functions but could also underlie region-specific sensitivities to microglial dysregulation and involvement in age-related neurodegeneration.

Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts.

Avian colibacillosis is caused by a group of pathogens designated avian pathogenic Escherichia coli (APEC). Despite being known for over a century, avian colibacillosis remains one of the major endemic diseases afflicting the poultry industry worldwide. Autologous bacterins provide limited serotype-specific protection, yet multiple serogroups are associated with disease, especially O1, O2 and O78 among many others. Experimental infection models have facilitated the identification of some key APEC virulence genes and have allowed testing of vaccine candidates. Well-recognized virulence factors include Type 1 (F1) and P (Pap/Prs) fimbriae for colonization, IbeA for invasion, iron acquisition systems, TraT and Iss for serum survival, K and O antigens for anti-phagocytic activity, and a temperature-sensitive haemagglutinin of imprecise function. Intriguingly, these factors do not occur universally among APEC, suggesting the presence of multiple alternative mechanisms mediating pathogenicity. The recent availability of the first complete APEC genome sequence can be expected to accelerate the identification of bacterial genes expressed during infection and required for virulence. High-throughput molecular approaches like signature-tagged transposon mutagenesis have already proved invaluable in revealing portfolios of genes expressed by pathogenic bacteria during infection, and this has enabled identification of APEC O2 factors required for septicaemia in the chicken model. Complimentary approaches, such as in vivo-induced antigen technology, exist to define the activities of APEC in vivo. In recent years, reverse vaccinology and immuno-proteomic approaches have also enabled identification of novel vaccine candidates in other bacterial pathogens. Collectively, such information provides the basis for the development or improvement of strategies to control APEC infections in the food-producing avian species.

A Burkholderia pseudomallei Type III Secreted Protein, BopE, Facilitates Bacterial Invasion of Epithelial Cells and Exhibits Guanine Nucleotide Exchange Factor Activity

We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2. Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion. Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for Cdc42 and Rac1 in vitro.

Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram‐negative facultative intracellular pathogen that enters and escapes from eukaryotic cells using the power of actin polymerization. We have identified a bacterial protein (BimA) that is required for the ability of B. pseudomallei to induce the formation of actin tails. BimA contains proline‐rich motifs and WH2‐like domains and shares limited homology at the C‐terminus with the Yersinia autosecreted adhesin YadA. BimA is located at the pole of the bacterial cell at which actin polymerization occurs and mutation of bimA abolished actin‐based motility of the pathogen in J774.2 cells. Transient expression of BimA in HeLa cells resulted in F‐actin clustering reminiscent of that seen on WASP overexpression. Antibody‐mediated clustering of a CD32 chimera in which the cytoplasmic domain was replaced with BimA resulted in localization of the chimera to the tips of F‐actin enriched membrane protrusions. We report that purified truncated BimA protein binds monomeric actin in a concentration‐dependent manner in cosedimentation assays and that BimA stimulates actin polymerization in vitro in a manner independent of the cellular Arp2/3 complex.

Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate

Amino acids are key carbon and energy sources for the asaccharolytic food‐borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen‐limited growth of C. jejuni in an aspA‐dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81‐176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.

Efa1 Influences Colonization of the Bovine Intestine by Shiga Toxin-Producing Escherichia coli Serotypes O5 and O111

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) comprises a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in animals and humans. Non-O157 STEC serotypes contain a gene (efa1) that mediates attachment to cultured epithelial cells. An almost-identical gene in enteropathogenic E. coli (lifA) encodes lymphostatin, which inhibits the proliferation of mitogen-activated lymphocytes and the synthesis of proinflammatory cytokines. We have investigated the role of the efa1 gene in colonization of 4- and 11-day-old conventional calves by STEC serotypes O5 and O111. Our findings show that Efa1 is required for efficient colonization of the bovine intestinal tract by STEC, since efa1 deletion and insertion mutants were shed in the feces in significantly lower numbers. In addition, efa1 mutations dramatically reduced the number of bacteria associated with the intestinal epithelium. Expression and secretion of locus for enterocyte effacement-encoded type III secreted proteins that are required for adhesion and AE-lesion formation were impaired by mutation of efa1 in STEC but not by mutation of lifA in enteropathogenic E. coli. However, STEC efa1 mutants retain the ability to nucleate filamentous actin under sites of bacterial attachment to cultured eukaryotic cells. Efa1 is only the second STEC factor shown to influence carriage of the bacteria in the bovine intestine. Our data may have implications for strategies to reduce the prevalence of STEC in cattle.

Molecular insights into farm animal and zoonotic Salmonella infections.

Salmonella enterica is a facultative intracellular pathogen of worldwide importance. Infections may present in a variety of ways, from asymptomatic colonization to inflammatory diarrhoea or typhoid fever depending on serovar- and host-specific factors. Human diarrhoeal infections are frequently acquired via the food chain and farm environment by virtue of the ability of selected non-typhoidal serovars to colonize the intestines of food-producing animals and contaminate the avian reproductive tract and egg. Colonization of reservoir hosts often occurs in the absence of clinical symptoms; however, some S. enterica serovars threaten animal health owing to their ability to cause acute enteritis or translocate from the intestines to other organs causing fever, septicaemia and abortion. Despite the availability of complete genome sequences of isolates representing several serovars, the molecular mechanisms underlying Salmonella colonization, pathogenesis and transmission in reservoir hosts remain ill-defined. Here we review current knowledge of the bacterial factors influencing colonization of food-producing animals by Salmonella and the basis of host range, differential virulence and zoonotic potential.