Sandra Muschiol

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3β to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re‐differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti‐chlamydial drugs.

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

BackgroundChlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (In nate P harmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.ResultsWe demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry.ConclusionINPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.

Identification of a family of effectors secreted by the type III secretion system that are conserved in pathogenic Chlamydiae.

ABSTRACT Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.

pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood–brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

Uptake of extracellular DNA: Competence induced pili in natural transformation of Streptococcus pneumoniae

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram-positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram-negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram-positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram-positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus-like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus-related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.

Human enteroendocrine cell responses to infection with Chlamydia trachomatis: a microarray study

BackgroundEnteroendocrine cells (EEC) are highly specialized cells producing signalling molecules vital to the normal functions of the gut. Recently, we showed altered protein distribution in Chlamydia infected EEC in vitro. The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model.MethodsTwo human EEC lines: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected using cultured C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. We used microarray analysis (Affymetrix GeneChip®) for studying changes in gene expression at different stages of infection.ResultsTwenty-four hours after active and persistent infection, 66 and 411 genes in LCC-18 and 68 and 170 genes in CNDT-2 cells, respectively showed mean expression ratios >2-fold compared to non-infected cells. These genes encoded factors regulating apoptosis, cell differentiation, transcription regulation, cytokine activity, amine biosynthesis and vesicular transport. We found significant differences in gene transcription levels between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) and in 5 genes related to endocrine function (GABARAPL1, GRIP1, DRD2, SYT5 and SYT7).ConclusionsInfected EEC cells exhibit cell-type specific patterns related to vesicular transport, secretion and neurotransmitters. EEC play a pivotal role in regulation of gut motility and an impairment of enteroendocrine function can contribute to motility disorders.

Infection of human enteroendocrine cells with Chlamydia trachomatis: a possible model for pathogenesis in irritable bowel syndrome

Background  Irritable bowel syndrome (IBS) is a widespread gastrointestinal disorder of unknown etiology. Recently, our group detected chlamydial antigens in enteroendocrine cells (EEC) of jejunum biopsies from patients with IBS. Impairment of EEC secretion upon Chlamydia infection might lead to disturbances of gut functions. We have therefore studied the interaction between Chlamydia and EEC in vitro.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae

Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These “competence pili” are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification

Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.Streptococcus pneumoniae evades the action of the complement system by expressing an immuno-protective polysaccharide capsule as well as Factor H-binding proteins. Here, Pathak et al. show that these two defence mechanisms are functionally and spatially coordinated on the bacterial cell surface.