Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer.

Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.

ß-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses.

The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct ß-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow-derived macrophages infected with Salmonella Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.

Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Delivery of cardiolipins to the Salmonella outer membrane is necessary for survival within host tissues and virulence.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that serves as a barrier to the environment. During infection, Gram-negative bacteria remodel their OM to promote survival and replication within host tissues. Salmonella rely on the PhoPQ two-component regulators to coordinate OM remodeling in response to environmental cues. In a screen for mediators of PhoPQ-regulated OM remodeling in Salmonella Typhimurium, we identified PbgA, a periplasmic domain-containing transmembrane protein, which binds cardiolipin glycerophospholipids near the inner membrane and promotes their PhoPQ-regulated trafficking to the OM. Purified-PbgA oligomers are tetrameric, and the periplasmic domain contains a globular region that binds to the OM in a PhoPQ-dependent manner. Thus, PbgA forms a complex that may bridge the envelope for regulated cardiolipin delivery. PbgA globular region-deleted mutant bacteria are severely attenuated for pathogenesis, suggesting that increased cardiolipin trafficking to the OM is necessary for Salmonella to survive within host tissues that activate PhoPQ.

Temporal and anatomical host resistance to chronic Salmonella infection is quantitatively dictated by Nramp1 and influenced by host genetic background.

The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2-3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg

A Salmonella typhimurium-translocated glycerophospholipid:cholesterol acyltransferase promotes virulence by binding to the RhoA protein switch regions.

Salmonella enterica serovar typhimurium translocates a glycerophospholipid:cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.

Identification of an unconventional E3 binding surface on the UbcH5 ~ Ub conjugate recognized by a pathogenic bacterial E3 ligase.

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ~ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ~ Ub(n)). Rapid generation of UbcH5 ~ Ub(n) may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation.

The Salmonella typhimurium type III secretion effector protein SifA is essential for inducing tubulation of the Salmonella phagosome and binds the mammalian kinesin-binding protein SKIP. Coexpression of SifA with the effector SseJ induced tubulation of mammalian cell endosomes, similar to that induced by Salmonella infection. Interestingly, GTP-bound RhoA, RhoB, and RhoC also induced endosomal tubulation when coexpressed with SseJ, indicating that SifA likely mimics or activates a RhoA family GTPase. The structure of SifA in complex with the PH domain of SKIP revealed that SifA has two distinct domains; the amino terminus binds SKIP, and the carboxyl terminus has a fold similar to SopE, a Salmonella effector with Rho GTPase guanine nucleotide exchange factor activity (GEF). Similar to GEFs, SifA interacted with GDP-bound RhoA, and purified SseJ and RhoA formed a protein complex, suggesting that SifA, SKIP, SseJ, and RhoA family GTPases cooperatively promote host membrane tubulation.

An inhibitor of gram-negative bacterial virulence protein secretion.

Bacterial virulence mechanisms are attractive targets for antibiotic development because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen of small molecule libraries and identified one compound, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and type II secretion-dependent functions, suggesting that its target could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.