Virulence-associated type III secretion systems in Gram-negative bacteria.

Virulence-associated bacterial type III secretion systems are multiprotein molecular machines that promote the pathogenicity of bacteria towards eukaryotic host cells. These machines form needle-like structures, named injectisomes, that span both bacterial and host membranes, forming a direct conduit for the delivery of bacterial proteins into host cells. Once within the host, these bacterial effector proteins are capable of manipulating a multitude of host cell functions. In recent years, the knowledge of assembly, structure and function of these machines has grown substantially and is presented and discussed in this review.

Sex Differences in Estrogen Receptor α and ß Levels and Activation Status in LPS-Stimulated Human Macrophages.

Immune function, inflammation, and atherosclerosis display sex differences and are influenced by 17ß-estradiol through estrogen receptors subtypes ERα and ERß. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERß and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex-specific way, the expression and the activation status of ERα and ERß in blood monocytes-derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERß, P-ERα, p38, and P-p38 expression by Western Blotting. In basal conditions, ERα and ERß were significantly higher in female MDMs than in male MDMs. LPS up-regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down-regulated ERß level only in female MDMs. p38 and P-p38 proteins, indicative of ERß activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERß and P-ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERß activation status. We identify a significant role of ERα in LPS-mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. J. Cell. Physiol. 232: 340-345, 2017. © 2016 Wiley Periodicals, Inc.

Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous system.

BACKGROUND: Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates. RESULTS: We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of ß-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells. CONCLUSIONS: Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.

Analysis of SPI-1 Dependent Type III Secretion and Injection Using a NanoLuc Luciferase-Based Assay.

Studies of bacterial protein secretion have relied on a variety of reporters that allow the tracking of secreted proteins. However, the lack of truly quantitative and highly sensitive reporters has hindered, in particular, the investigation of the kinetics of protein secretion. In this chapter, we describe a luminescence-based assay using NanoLuc luciferase to analyse secretion and injection into host cells of type III secretion (T3S) substrates encoded on Salmonella pathogenicity island-1 (SPI-1). This method has a very high sensitivity and high signal-to-noise ratio. Moreover, the simplicity of the protocol and the rapid determination and quantification of the luminescence makes it ideal for the monitoring of the kinetics of secretion but also convenient for high-throughput screenings. The protocols presented here include (1) Salmonella SPI-1 secretion assay, where the T3S substrates-NanoLuc fusions are detected by luminometry in the bacterial supernatant, and (2) Salmonella injection assays, using the split-Nanoluc (HiBiT/LgBiT) to monitor the injection of T3S substrates-HiBiT fusions into the host cells stably expressing LgBiT.

The Type III Secretion Effector CteG Mediates Host Cell Lytic Exit of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.

The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells.

Chlamydia trachomatis causes genital and ocular infections in humans. This bacterial pathogen multiplies exclusively within host cells in a characteristic vacuole (inclusion) and delivers proteins such as inclusion membrane proteins (Incs) into the host cell. Here, we identified CT006 as a novel C. trachomatis protein that when expressed ectopically eukaryotic cells can associate with lipid droplets (LDs). A screen using Saccharomyces cerevisiae identified two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. Ectopic expression in yeast and mammalian cells of genes encoding different fragments of CT006 revealed tropism for the endoplasmic reticulum and LDs. We identified a LD-targeting region within the first 88 amino acid residues of CT006, and positively charged residues important for this targeting. Comparing with the parental wild-type strain, cells infected by a newly generated C. trachomatis strain overproducing CT006 with a double hemagglutinin tag showed a slight increase in the area occupied by LDs within the inclusion region. However, we could not correlate this effect with the LD-targeting regions within CT006. We further showed that both the amino and carboxy-terminal regions of CT006, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, supporting their availability to interact with host cell targets. Altogether, our data suggest that CT006 might participate in the interaction of LDs with C. trachomatis inclusions.

CteG is a Chlamydia trachomatis effector protein that associates with the Golgi complex of infected host cells.

Chlamydia trachomatis is a bacterial pathogen causing ocular and genital infections in humans. C. trachomatis multiplies exclusively inside host cells within a characteristic vacuole, from where it manipulates host cells by injecting them with type III secretion effector proteins. Here, we identified CteG as the first C. trachomatis effector associated with the Golgi. For this, C. trachomatis strains expressing candidate effectors fused to a double hemagglutinin (2HA) tag were constructed. Then, among these strains, immunofluorescence microscopy revealed that CteG-2HA was delivered into the cytoplasm of infected cells. Between 16-20 h post-infection, CteG-2HA mostly associated with the Golgi; however, CteG-2HA also appeared at the host cell plasma membrane, and at 30 or 40 h post-infection this was its predominant localization. This change in the main localization of CteG-2HA was independent of intact microfilaments or microtubules. Ectopic expression of different regions of CteG (656 amino acid residues) in uninfected cells revealed that its first 100 residues contain a Golgi targeting region. Although a C. trachomatis cteG mutant did not display a defect in intracellular multiplication, CteG induced a vacuolar protein sorting defect when expressed in Saccharomyces cerevisiae. This suggested that CteG might function by subverting host cell vesicular transport.

The Human Centrosomal Protein CCDC146 Binds Chlamydia trachomatis Inclusion Membrane Protein CT288 and Is Recruited to the Periphery of the Chlamydia-Containing Vacuole.

Chlamydia trachomatis is an obligate intracellular human pathogen causing mainly ocular and genital infections of significant clinical and public health impact. C. trachomatis multiplies intracellularly in a membrane bound vacuole, known as inclusion. Both extracellularly and from within the inclusion, C. trachomatis uses a type III secretion system to deliver several effector proteins into the cytoplasm of host cells. A large proportion of these effectors, the inclusion membrane (Inc) proteins, are exposed to the host cell cytosol but possess a characteristic hydrophobic domain mediating their insertion in the inclusion membrane. By yeast two-hybrid, we found that C. trachomatis Inc CT288 interacts with the human centrosomal protein CCDC146 (coiled-coil domain-containing protein 146). The interaction was also detected by co-immunoprecipitation in mammalian cells either ectopically expressing CCDC146 and CT288 or ectopically expressing CCDC146 and infected by a C. trachomatis strain expressing epitope-tagged and inclusion membrane-localized CT288. In uninfected mammalian cells, ectopically expressed full-length CCDC146 (955 amino acid residues) localized at the centrosome; but in cells infected by wild-type C. trachomatis, its centrosomal localization was less evident and CCDC146 accumulated around the inclusion. Recruitment of CCDC146 to the inclusion periphery did not require intact host Golgi, microtubules or microfilaments, but was dependent on chlamydial protein synthesis. Full-length CCDC146 also accumulated at the periphery of the inclusion in cells infected by a C. trachomatis ct288 mutant; however, a C-terminal fragment of CCDC146 (residues 692-955), which interacts with CT288, showed differences in localization at the periphery of the inclusion in cells infected by wild-type or ct288 mutant C. trachomatis. This suggests a model in which chlamydial proteins other than CT288 recruit CCDC146 to the periphery of the inclusion, where the CT288-CCDC146 interaction might contribute to modulate the function of this host protein.

Effector Translocation Assay: Differential Solubilization.

The identification of effector proteins delivered into mammalian host cells by bacterial pathogens possessing syringelike nanomachines is an important step toward understanding the mechanisms underlying the virulence of these pathogens. In this chapter, we describe a method based on mammalian tissue culture infection models where incubation with a nonionic detergent (Triton X-100) enables solubilization of host cell membranes but not of bacterial membranes. This allows the isolation of a Triton-soluble fraction lacking bacteria but enriched in proteins present in the host cell cytoplasm and plasma membrane. Using appropriate controls, this fraction can be probed by immunoblotting for the presence of bacterial effector proteins delivered into host cells.

The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion.

Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20-26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.

Identification of novel type III secretion chaperone-substrate complexes of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipitation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.