Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of ...infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.
Campylobacteriosis is the leading foodborne bacterial diarrheal illness in many countries, with up to 80% of human cases attributed to the avian reservoir. The only control strategies currently ...available are stringent on-farm biosecurity and carcass treatments. Heritable differences in the resistance of chicken lines to
colonization have been reported and resistance-associated quantitative trait loci are emerging, although their impact on colonization appears modest. Recent studies indicated a protective role of the microbiota against colonization by
in chickens. Furthermore, in murine models, differences in resistance to bacterial infections can be partially transferred between lines by transplantation of gut microbiota. In this study, we investigated whether heritable differences in colonization of inbred chicken lines by
are associated with differences in cecal microbiota. We performed homologous and heterologous cecal microbiota transplants between line 6
(resistant) and line N (susceptible) by orally administering cecal contents collected from 3-week-old donors to day-of-hatch chicks. Recipient birds were challenged (day 21) with
11168H. In birds given homologous microbiota, the differential resistance of lines to
colonization was reproduced. Contrary to our hypothesis, transfer of cecal microbiota from line 6
to line N significantly increased
colonization. No significant difference in the overall composition of the cecal microbial communities of the two lines was identified, although line-specific differences for specific operational taxonomic units were identified. Our data suggest that while heritable differences in avian resistance to
colonization exist, these are not explained by significant variation in the cecal microbiota.
is a leading cause of foodborne diarrheal disease worldwide. Poultry are a key source of human infections, but there are currently few effective measures against
in poultry during production. One option to control
may be to alter the composition of microbial communities in the avian intestines by introducing beneficial bacteria, which exclude the harmful ones. We previously described two inbred chicken lines which differ in resistance to intestinal colonization by
Here, we investigated the composition of the microbial communities in the gut of these lines and whether transferring gut bacteria between the resistant and susceptible lines alters their resistance to
No major differences in microbial populations were found, and resistance or susceptibility to colonization was not conferred by transferring gut bacteria between lines. The data suggest that gut microbiota did not play a role in resistance to
colonization, at least in the lines used.
MHCY is a candidate region for influencing immune responses in chickens. MHCY contains multiple specialized, polymorphic MHC class I loci along with loci belonging to 4 additional gene families. In ...this study, MHCY haplotypes were tested for association with cecal colonization after Campylobacter jejuni infection of a backcross (Line 61 × Line N) × Line N population derived from 2 White Leghorn research lines, Line 61 and Line N, that were previously shown to exhibit heritable differences in colonization. Samples were obtained for 51 birds challenged with 108 CFU Campylobacter jejuni at 3 wk of age. Viable C. jejuni in the ceca were enumerated 5 d postinfection and counts were log-transformed for analysis. Birds were assigned to either low or high colonization groups based on the individual count being below or above the mean bacterial count for all birds. The mean bacterial count of the low infection group differed significantly from the high infection group. Sex and MHCB haplotype had similar distributions within the 2 groups. Overall, 7 MHCY haplotypes were found to be segregating. Two were significantly associated with C. jejuni colonization. MHCY Y18 was associated with low colonization (P = 3.00 × 10−5); whereas MHCY Y11a was associated with high colonization (P = 0.008). The MHCY haplotype impacted the mean bacterial count among all birds with MHCY Y18 having the lowest bacterial count compared with MHCY Y11a and all other MHCY (Y5, Y7, Y8, Y11b, and Y11c) haplotypes. These findings support further investigation of the contribution of chicken MHCY in resistance to Campylobacter colonization.
Salmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of ...virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII
macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.
Avian pathogenic
(APEC) cause severe respiratory and systemic disease in chickens, commonly termed colibacillosis. Early immune responses after initial infection are highly important for the outcome ...of the infection. In this study, the early interactions between
-expressing APEC strains of serotypes O1:K1:H7 and O2:K1:H5 and phagocytic cells in the lung of
-reporter transgenic chickens were investigated.
-reporter transgenic chickens express fluorescent protein under the control of elements of the
promoter and enhancer, such that cells of the myeloid lineage can be visualized
and sorted. Chickens were separately inoculated with APEC strains expressing
and culled 6 h post-infection. Flow cytometric analysis was performed to phenotype and sort the cells that harbored bacteria in the lung, and the response of the sorted cells was defined by transcriptomic analysis. Both APEC strains were mainly detected in
-transgene
(
-tg
) and
-tg
MHC II
MRC1L-B
cells and low numbers of APEC were detected in
-tg
MHC II
MRC1L-B
cells. Transcriptomic and flow cytometric analysis identified the APEC
-tg
and
-tg
cells as heterophils and the APEC
-tg
cells as macrophages and dendritic cells. Both APEC strains induced strong inflammatory responses, however in both
-tg
and
-tg
cells, many immune related pathways were repressed to a greater extent or less activated in birds inoculated with APEC O2-
compared to APEC O1-
inoculated birds. Comparison of the immune pathways revealed the aryl hydrocarbon receptor (
) pathway,
and
signaling, heterophil recruitment pathways and the acute phase response, are modulated particularly post-APEC O2-
inoculation. In contrast to
data, APEC O2-
was more invasive in
-tg
cells
than APEC O1-
and had higher survival rates for up to 6 h post-infection. Our data indicate significant differences in the responses induced by APEC strains of prevalent serotypes, with important implications for the design and interpretation of future studies. Moreover, we show that bacterial invasion and survival in phagocyte populations
is not predictive of events in the chicken lung.
Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of ...cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.
Lymphostatin is a virulence factor of enteropathogenic
E. coli
(EPEC) and non-O157 serogroup enterohaemorrhagic
E. coli
. Previous studies using whole-cell lysates of EPEC showed that lymphostatin ...inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4
+
and CD8
+
T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4
+
and CD8
+
T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27
kip1
, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.
Nanoscale membrane curvature is a common feature in cell biology required for functions such as endocytosis, exocytosis and cell migration. These processes require the cytoskeleton to exert forces on ...the membrane to deform it. Cytosolic proteins contain specific motifs which bind to the membrane, connecting it to the internal cytoskeletal machinery. These motifs often bind charged phosphatidylinositol phosphate lipids present in the cell membrane which play significant roles in signaling. These lipids are important for membrane deforming processes, such as endocytosis, but much remains unknown about their role in the sensing of membrane nanocurvature by protein domains. Using coarse-grained molecular dynamics simulations, we investigated the interaction of a model curvature active protein domain, the epsin N-terminal homology domain (ENTH), with curved lipid membranes. The combination of anionic lipids (phosphatidylinositol 4,5-bisphosphate and phosphatidylserine) within the membrane, protein backbone flexibility, and structural changes within the domain were found to affect the domain’s ability to sense, bind, and localize with nanoscale precision at curved membrane regions. The findings suggest that the ENTH domain can sense membrane curvature without the presence of its terminal amphipathic α helix via another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.
So far six susceptibility loci for renal cell carcinoma (RCC) have been discovered by genome-wide association studies (GWAS). To identify additional RCC common risk loci, we performed a meta-analysis ...of published GWAS (totalling 2,215 cases and 8,566 controls of Western-European background) with imputation using 1000 Genomes Project and UK10K Project data as reference panels and followed up the most significant association signals 22 single nucleotide polymorphisms (SNPs) and 3 indels in eight genomic regions in 383 cases and 2,189 controls from The Cancer Genome Atlas (TCGA). A combined analysis identified a promising susceptibility locus mapping to 1q24.1 marked by the imputed SNP rs3845536 (Pcombined =2.30x10-8). Specifically, the signal maps to intron 4 of the ALDH9A1 gene (aldehyde dehydrogenase 9 family, member A1). We further evaluated this potential signal in 2,461 cases and 5,081 controls from the International Agency for Research on Cancer (IARC) GWAS of RCC cases and controls from multiple European regions. In contrast to earlier findings no association was shown in the IARC series (P=0.94; Pcombined =2.73x10-5). While variation at 1q24.1 represents a potential risk locus for RCC, future replication analyses are required to substantiate our observation.
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