Comprehensive assignment of roles for Salmonella typhimurium genes in intestinal colonization of food-producing animals.

Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype-genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals.

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.

Identification of a BTV-Strain-Specific Single Gene That Increases Culicoides Vector Infection Rate.

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-14S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-41S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-14S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.

An Early Block in the Replication of the Atypical Bluetongue Virus Serotype 26 in Culicoides Cells Is Determined by Its Capsid Proteins.

Arboviruses such as bluetongue virus (BTV) replicate in arthropod vectors involved in their transmission between susceptible vertebrate-hosts. The "classical" BTV strains infect and replicate effectively in cells of their insect-vectors (Culicoides biting-midges), as well as in those of their mammalian-hosts (ruminants). However, in the last decade, some "atypical" BTV strains, belonging to additional serotypes (e.g., BTV-26), have been found to replicate efficiently only in mammalian cells, while their replication is severely restricted in Culicoides cells. Importantly, there is evidence that these atypical BTV are transmitted by direct-contact between their mammalian hosts. Here, the viral determinants and mechanisms restricting viral replication in Culicoides were investigated using a classical BTV-1, an "atypical" BTV-26 and a BTV-1/BTV-26 reassortant virus, derived by reverse genetics. Viruses containing the capsid of BTV-26 showed a reduced ability to attach to Culicoides cells, blocking early steps of the replication cycle, while attachment and replication in mammalian cells was not restricted. The replication of BTV-26 was also severely reduced in other arthropod cells, derived from mosquitoes or ticks. The data presented identifies mechanisms and potential barriers to infection and transmission by the newly emerged "atypical" BTV strains in Culicoides.

Norepinephrine augments Salmonella enterica-induced enteritis in a manner associated with increased net replication but independent of the putative adrenergic sensor kinases QseC and QseE.

Stress has long been correlated with susceptibility to microbial infection. One explanation for this phenomenon is the ability of pathogens to sense and respond to host stress-related catecholamines, such as norepinephrine (NE). In Gram-negative enteric pathogens, it has been proposed that NE may facilitate growth by mediating iron supply, or it may alter gene expression by activating adrenergic sensor kinases. The aim of this work was to investigate the relative importance of these processes in a model in which NE alters the outcome of Salmonella enterica serovar Typhimurium infection. A bovine ligated ileal loop model was used to study the effect of NE on enteritis induced by S. Typhimurium and on the bacterial in vivo replication rate. Mutants lacking putative adrenergic receptor genes were assessed in the loop model, in a calf intestinal colonization model, and in vitro. S. Typhimurium-induced enteritis was significantly enhanced by addition of 5 mM NE. This effect was associated with increased net bacterial replication in the same model. Exogenous ferric iron also stimulated bacterial replication in the medium used but not transcription of enteritis-associated loci. The putative adrenergic sensors QseC and QseE were not required for NE-enhanced enteritis, intestinal colonization of calves, or NE-dependent growth in iron-restricted medium and did not influence expression or secretion of enteritis-associated virulence factors. Our findings support a role for stress-related catecholamines in modulating the virulence of enteric bacterial pathogens in vivo but suggest that bacterial adrenergic sensors may not be the vital link in such interkingdom signaling in Salmonella.

Role of two-component sensory systems of Salmonella enterica serovar Dublin in the pathogenesis of systemic salmonellosis in cattle.

Salmonella enterica serovar Dublin (S. Dublin) is associated with enteritis, typhoid and abortion in cattle. Infections are acquired by the oral route, and the bacteria transit through varied anatomical and cellular niches to elicit systemic disease. S. Dublin must therefore sense and respond to diverse extrinsic stimuli to control gene expression in a spatial and temporal manner. Two-component systems (TCSs) play key roles in such processes, and typically contain a membrane-associated sensor kinase (SK) that modifies a cognate response regulator. Analysis of the genome sequence of S. Dublin identified 31 conserved SK genes. Each SK gene was separately disrupted by lambda Red recombinase-mediated insertion of transposons harbouring unique sequence tags. Calves were challenged with a pool of the mutants together with control strains of defined virulence by the oral and intravenous routes. Quantification of tagged mutants in output pools derived from various tissues and cannulated lymphatic vessels allowed the assignment of spatial roles for each SK following oral inoculation or when the intestinal barrier was bypassed by intravenous delivery. Mutant phenotypes were also assigned in cultured intestinal epithelial cells. Mutants with insertions in barA, envZ, phoQ, ssrA or qseC were significantly negatively selected at all enteric and systemic sites sampled after oral dosing. Mutants lacking baeS, dpiB or citA were negatively selected at some but not all sites. After intravenous inoculation, only barA and phoQ mutants were significantly under-represented at systemic sites. The novel role of baeS in intestinal colonization was confirmed by oral co-infection studies, with a mutant exhibiting modest but significant attenuation at a number of enteric sites. This is the first systematic analysis of the role of all Salmonella TCSs in a highly relevant model of enteric fever. Spatial roles were assigned to eight S. Dublin SKs, but most were not essential for intestinal or systemic infection of the target host.

Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle.

Salmonella enterica serovar Dublin is a host-restricted serovar associated with typhoidal disease in cattle. In contrast, the fowl-associated serovar S. enterica serovar Gallinarum is avirulent in calves, yet it invades ileal mucosa and induces enteritis at levels comparable to those induced by S. enterica serovar Dublin. Suppression subtractive hybridization was employed to identify S. enterica serovar Dublin strain SD3246 genes absent from S. enterica serovar Gallinarum strain SG9. Forty-one S. enterica serovar Dublin fragments were cloned and sequenced. Among these, 24 mobile-element-associated genes were identified, and 12 clones exhibited similarity with sequences of known or predicted function in other serovars. Three S. enterica serovar Dublin-specific regions were homologous to regions from the genome of Enterobacter sp. strain 638. Sequencing of fragments adjacent to these three sequences revealed the presence of a 21-kb genomic island, designated S. enterica serovar Dublin island 1 (SDI-1). PCR analysis and Southern blotting showed that SDI-1 is highly conserved within S. enterica serovar Dublin isolates but rarely found in other serovars. To probe the role of genes identified by subtractive hybridization in vivo, 24 signature-tagged S. enterica serovar Dublin SD3246 mutants lacking loci not present in Salmonella serovar Gallinarum SG9 were created and screened by oral challenge of cattle. Though attenuation of tagged SG9 and SD3246 Salmonella pathogenicity island-1 (SPI-1) and SPI-2 mutant strains was detected, no obvious defects of these 24 mutants were detected. Subsequently, a DeltaSDI-1 mutant was found to exhibit weak but significant attenuation compared with the parent strain in coinfection of calves. SDI-1 mutation did not impair invasion, intramacrophage survival, or virulence in mice, implying that SDI-1 does not influence fitness per se and may act in a host-specific manner.

Multilocus-sequence typing analysis reveals similar populations of Streptococcus uberis are responsible for bovine intramammary infections of short and long duration.

Multilocus-sequence typing (MLST) was used to analyse Streptococcus uberis isolates from a single herd associated with long duration (50-260 days) and rapidly cleared (less than 1 month) bovine intramammary infections to determine whether the bacterial type had any impact on the duration of infection. Most chronic infections (24 of 33) were due to continuous infection of the mammary quarter with the same sequence type, and infections were found to persist for many months. The remaining quarters were re-infected with a different sequence type within a single lactation. No particular sequence type or clonal complex (lineage) was associated with persisting infections, indicating that the outcome of intramammary infections with S. uberis is more likely to be dependent on host factors than on inter-strain differences. Analysis of these strains alongside others obtained from the same herd at a later date revealed the shift in the predominant genotypes with time.

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells).

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional 'types' have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.

6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs.

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A DeltaqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the DeltaqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.

Histidine Residues at the Active Site of the Pasteurella multocida Toxin.

We have investigated histidine residues near the active site of the mitogenic Pasteurella multocida toxin. Mutation of H1202 or H1228 had little effect, while the effect of mutation on H1223 depended on the amino acid substituted. Mutation of H1205 caused complete loss of activity, indicating its importance in PMT activity.

Systemic translocation of Salmonella enterica serovar Dublin in cattle occurs predominantly via efferent lymphatics in a cell-free niche and requires type III secretion system 1 (T3SS-1) but not T3SS-2.

Salmonella enterica is an important diarrheal pathogen, and infections may involve severe systemic sequelae depending on serovar- and host-specific factors. The molecular mechanisms underlying translocation of host-restricted and -specific serovars of S. enterica from the intestines to distal organs are ill defined. By surgical cannulation of lymph and blood vessels draining the distal ileum in cattle, S. enterica serovar Dublin was observed to translocate predominantly via mesenteric lymph nodes to efferent lymphatics in a manner that correlates with systemic virulence, since the fowl typhoid-associated serovar Gallinarum translocated at a significantly lower level. While both S. enterica serovars Dublin and Gallinarum were intracellular while in the intestinal mucosa and associated with major histocompatibility complex class II-positive cells, the bacteria were predominantly extracellular within efferent lymph. Screening of a library of signature-tagged serovar Dublin mutants following oral inoculation of calves defined the role of 36 virulence-associated loci in enteric and systemic phases of infection. The number and proportion of tagged clones reaching the liver and spleen early after oral infection were identical to the values in efferent lymph, implying that this may be a relevant mode of dissemination. Coinfection studies confirmed that lymphatic translocation requires the function of type III secretion system 1 (T3SS-1) but, remarkably, not T3SS-2. This is the first description of the mode and genetics of systemic translocation of serovar Dublin in its natural host.

First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology.

Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis throughout the world. Several procedures to differentiate S. uberis isolates have been proposed. However, all are prone to interlaboratory variation, and none is suitable for the description of the population structure. We describe here the development of a multilocus sequence typing (MLST) scheme for S. uberis to help address these issues. The sequences of seven housekeeping gene fragments from each of 160 United Kingdom milk isolates of S. uberis were determined. Between 5 and 17 alleles were obtained per locus, giving the potential to discriminate between 1.3 x 10(7) sequence types. In this study, 57 sequence types (STs) were identified. Statistical comparisons between the maximum-likelihood trees constructed by using the seven housekeeping gene fragments showed that the congruence was no better than that between each tree and trees of random topology, indicating there had been significant recombination within these loci. The population contained one major lineage (designated the ST-5 complex). This dominated the population, containing 24 STs and representing 112 isolates. The other 33 STs were not assigned to any clonal complex. All of the isolates in the ST-5 lineage carried hasA, a gene that is essential for capsule production. There was no clear association between ST or clonal complex and disease. The S. uberis MLST system offers researchers a valuable tool that allows further investigation of the population biology of this organism and insights into the epidemiology of this disease on a global scale.

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom.

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.

Pasteurella multocida toxin facilitates inositol phosphate formation by bombesin through tyrosine phosphorylation of G alpha q.

The intracellularly acting Pasteurella multocida toxin (PMT) is a potent mitogen that stimulates Gq-dependent formation of inositol trisphosphate. We show that PMT, a nontoxic mutant of PMT (PMTC1165S), and bombesin each stimulate time-dependent phosphorylation of G alpha q at tyrosine 349. Although PMT and PMTC1165S each cause phosphorylation of G alpha q, only the wild-type toxin activates Gq. Pretreatment of cells with wild-type or mutant PMT potentiated the formation of inositol phosphates stimulated by bombesin equally. These data show that PMT potentiates bombesin receptor signaling through tyrosine phosphorylation of Gq and distinguishes between the two proposed models of Gq activation, showing that tyrosine phosphorylation is not linked to receptor uncoupling.

The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation.

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.

Role of the dermonecrotic toxin of Bordetella bronchiseptica in the pathogenesis of respiratory disease in swine.

Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT(+) parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.