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ItemIntracellular demography and the dynamics of Salmonella enterica infections.(Public Library of Science (PLoS), 2006-10)An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.
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ItemDexamethasone modulates Salmonella enterica serovar Typhimurium infection in vivo independently of the glucocorticoid-inducible protein annexin-A1.(Oxford University Press (OUP), 2008-12)Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice.
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ItemGenomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens.(Public Library of Science (PLoS), 2009-03)The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A(2) toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci.
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ItemPost-assembly modification of Bordetella bronchiseptica O polysaccharide by a novel periplasmic enzyme encoded by wbmE.(Elsevier BV, 2009-01-16)Bordetella bronchiseptica is a pathogen of humans and animals that colonizes the respiratory tract. It produces a lipopolysaccharide O antigen that contains a homopolymer of 2,3-dideoxy-2,3-diacetamido-L-galacturonic acid (L-GalNAc3NAcA). Some of these sugars are found in the uronamide form (L-GalNAc3NAcAN), and there is no discernible pattern in the distribution of amides along the chain. A B. bronchiseptica wbmE mutant expresses an O polysaccharide unusually rich in uronamides. The WbmE protein localizes to the periplasm and catalyzes the deamidation of uronamide-rich O chains in lipopolysaccharide purified from the mutant, to attain a wild-type uronamide/uronic acid ratio. WbmE is a member of the papain-like transglutaminase superfamily, and this categorization is consistent with a deamidase role. The periplasmic location of WbmE and its acceptance of complete lipopolysaccharide as substrate indicate that it operates at a late stage in lipopolysaccharide biosynthesis, after polymerization and export of the O chain from the cytoplasm. This is the first report of such a modification of O antigen after assembly. The expression of wbmE is controlled by the Bordetella virulence gene two-component regulatory system, BvgAS, suggesting that this deamidation is a novel mechanism by which these bacteria modify their cell surface charge in response to environmental stimuli.
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ItemEvidence for niche adaptation in the genome of the bovine pathogen Streptococcus uberis.(Springer Science and Business Media LLC, 2009-01-28)BACKGROUND: Streptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen. RESULTS: The genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified. CONCLUSION: S. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.
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ItemAn oral recombinant vaccine in dogs against Echinococcus granulosus, the causative agent of human hydatid disease: a pilot study.(Public Library of Science (PLoS), 2008-01-16)Dogs are the main source of human cystic echinococcosis. An oral vaccine would be an important contribution to control programs in endemic countries. We conducted two parallel experimental trials in Morocco and Tunisia of a new oral vaccine candidate against Echinococcus granulosus in 28 dogs. The vaccine was prepared using two recombinant proteins from adult worms, a tropomyosin (EgTrp) and a fibrillar protein similar to paramyosin (EgA31), cloned and expressed in a live attenuated strain of Salmonella enterica serovar typhimurium.In each country, five dogs were vaccinated with the associated EgA31 and EgTrp; three dogs received only the vector Salmonella; and six dogs were used as different controls. The vaccinated dogs received two oral doses of the vaccine 21 d apart, and were challenged 20 d later with 75,000 living protoscoleces. The controls were challenged under the same conditions. All dogs were sacrificed 26-29 d postchallenge, before the appearance of eggs, for safety reasons.We studied the histological responses to both the vaccine and control at the level of the duodenum, the natural localization of the cestode. Here we show a significant decrease of parasite burden in vaccinated dogs (70% to 80%) and a slower development rate in all remaining worms. The Salmonella vaccine EgA31-EgTrp demonstrated a high efficacy against E. granulosus promoting its potential role in reducing transmission to humans and animals.
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ItemModelling within-host spatiotemporal dynamics of invasive bacterial disease.(Public Library of Science (PLoS), 2008-04-08)Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics.
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ItemRapid evolution of virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis.(Public Library of Science (PLoS), 2009-07-15)BACKGROUND: Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, approximately 40% of the approximately 2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three approximately 90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. CONCLUSIONS/SIGNIFICANCE: The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.