Biology subjects

Sixt, B. S.

Publications and source records attributed to Sixt, B. S..

5 recordsLinked to original sources

bioRxiv2024

A genome-wide genetic screen identified targets for destabilizing the parasitophorous vacuole of Chlamydia trachomatis

The bacterial pathogen Chlamydia trachomatis employs the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. While conducting a CRISPR knock-out screen for genes contributing to this response, we uncovered a mutant deficient for CpoS to display a markedly increased reliance on host cellular ceramide synthesis, compensating for its diminished ability to acquire sphingolipids via modulating membrane trafficking. Employing the power of the just recently established molecular genetic toolbox for Chlamydia, we developed an innovative microscopic reporter system that revealed the mutant to thrive in unstable parasitophorous vacuoles (inclusions), characterized initially by the release of individual bacteria from otherwise intact-appearing vacuoles. CpoS-deficient inclusions were further destabilized by disruptions in ceramide synthesis, while supplementation of sphingoid bases stabilized them, also preventing the defensive host cell death response. Notably, early inclusion destabilization, achieved by simultaneous disruption of two transport routes, caused infection clearance without damaging the host cells. Overall, this study highlights the inclusions role as a refuge, demonstrates CpoS to maintain inclusion integrity by ensuring sphingolipid supply, and provides directions for a future therapeutic exploitation. SIGNIFICANCEA wide range of clinically significant microbes evolved to hide from the intrinsic defenses of their host cells by thriving within membrane-enclosed pathogen-containing vacuoles. This raises the intriguing possibility that such vacuoles could be targeted therapeutically. The bacterial pathogen Chlamydia trachomatis could be an exceptionally well-suited target for such innovative medicines given its medical importance and strict dependence on host cells. However, progress has been stalled by the lack of sensitive tools for detecting inclusion damage. Here, we resolved this major technical roadblock and uncovered the pathogen to employ the secreted effector CpoS, a modulator of membrane trafficking, to stabilize its vacuole by ensuring adequate sphingolipid supply. These methodological advances and mechanistic insights should promote the development of vacuole-destabilizing therapeutics.

microbiology↗

bioRxiv2023

A multi-strategy antimicrobial discovery approach reveals new ways to combat Chlamydia

While the excessive use of broad-spectrum antibiotics is a major driver of the global antibiotic resistance crisis, more selective therapies remain unavailable for the majority of bacterial pathogens. This includes Chlamydia spp., which cause millions of urogenital, ocular, and respiratory infections each year. We here report major conceptual additions to the available toolkit for antichlamydial discovery, including both experimental and computational approaches. Moreover, we report the to date most comprehensive search of the chemical space for novel antichlamydial activities, which identified over sixty compounds that are chemically diverse, structurally different from known antibiotics, non-toxic to human cells, and highly potent in blocking Chlamydia growth. While some compounds caused a reversible block in Chlamydia development, others could eradicate both established and persistent infections in a bactericidal manner. The most potent antichlamydials displayed compelling selectivity, some also synergies with clinically used antibiotics, as well as interactions profiles enabling predictions of molecular modes of action. Moreover, one compound displayed reduced antichlamydial efficacy in autophagy-deficient cells, suggesting a host- targeted activity. Altogether, we suggest that these novel antichlamydials could serve as tools for advancing our understanding of Chlamydia biology and as chemical starting points for developing more sustainable therapeutics for one of the most successful groups of intracellular pathogens.

microbiology↗

bioRxiv2023

The acetylase activity of Cdu1 protects Chlamydia effectors from degradation to regulate bacterial exit from infected cells

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen Chlamydia trachomatis secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins. Here we determined that Cdu1s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1 s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

microbiology↗

bioRxiv2022

Ecology and evolution of chlamydial symbionts of arthropods

The phylum Chlamydiae consists of obligate intracellular bacteria including major human pathogens and diverse environmental representatives. Here we investigated the Rhabdochlamydiaceae, which is predicted to be the largest and most diverse chlamydial family, with the few described members known to infect arthropod hosts. Using published 16S rRNA gene sequence data we identified at least 388 genus-level lineages containing about 14 051 putative species within this family. We show that rhabdochlamydiae are mainly found in freshwater and soil environments, suggesting the existence of diverse, yet unknown hosts. Next, we used a comprehensive genome dataset including metagenome assembled genomes classified as members of the family Rhabdochlamydiaceae, and we added novel complete genome sequences of Rhabdochlamydia porcellionis infecting the woodlouse Porcellio scaber, and of Candidatus R. oedothoracis associated with the linyphiid dwarf spider Oedothorax gibbosus. Comparative analysis of basic genome features and gene content with reference genomes of well-studied chlamydial families with known host ranges, namely Parachlamydiaceae (protist hosts) and Chlamydiaceae (human and other vertebrate hosts) suggested distinct niches for members of the Rhabdochlamydiaceae. We propose that members of the family represent intermediate stages of adaptation of chlamydiae from protists to vertebrate hosts. Within the genus Rhabdochlamydia, pronounced genome size reduction could be observed (1.49-1.93 Mb). The abundance and genomic distribution of transposases suggests transposable element expansion and subsequent gene inactivation as a mechanism of genome streamlining during adaptation to new hosts. This type of genome reduction has never been described before for any member of the phylum Chlamydiae. This study provides new insights into the molecular ecology, genomic diversity, and evolution of representatives of one of the most divergent chlamydial families.

genomics↗

bioRxiv2022

The Chlamydia protein CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35

The obligate intracellular bacterium Chlamydia trachomatis inserts into the membrane of its vacuole (the inclusion) a family of poorly characterized Inc proteins. While the Inc CpoS was recently revealed as a critical suppressor of host cellular immune surveillance, the underlying mechanism remained unknown. By complementing a cpoS mutant with modified variants of CpoS, we found that CpoS blocks distinct cellular defense responses through distinct mechanisms. Specifically, we show that the ability of CpoS to interact with Rab GTPases is not only instrumental to its ability to mediate lipid transport to the inclusion, but also key to CpoS-mediated inhibition of type I interferon responses. Indeed, depletion of Rab35 can phenocopy the respective defect of the cpoS mutant. Unexpectedly, we found that CpoS is also essential for the formation of inclusion microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. Overall, our findings highlight the modulation of membrane trafficking as a pathogenic immune evasion strategy and the role of Inc-Inc interactions in shaping the inclusion microenvironment.

microbiology↗