Our understanding of how the obligate intracellular bacterial pathogen
reprograms the function of infected cells in the upper genital tract is largely based on observations made in cell culture with ...transformed epithelial cell lines. Here, we describe a primary organoid system derived from endometrial tissue to recapitulate epithelial cell diversity, polarity and ensuing responses to
infection. Using high-resolution and time-lapse microscopy, we catalog the infection process in organoids from invasion to egress, including the reorganization of the cytoskeleton and positioning of intracellular organelles. We show this model is amenable to screening
mutants for defects in the fusion of pathogenic vacuoles, the recruitment of intracellular organelles and inhibition of cell death. Moreover, we reconstructed a primary immune cell response by co-culturing infected organoids with neutrophils, and determined that effectors like CPAF (also known as CT858) and TepP (also known as CT875) limit the recruitment of neutrophils to infected organoids. Collectively, our model can be applied to study the cell biology of
infections in three-dimensional structures that better reflect the diversity of cell types and polarity encountered by
in their animal hosts.
Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate ...with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with "non-self" PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on "self" organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of "self" IRGM proteins from these structures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The
comprise a group of highly adapted bacterial pathogens sharing a unique intracellular lifestyle. Three
species are pathogenic to humans:
, and
.
is the leading bacterial cause of ...sexually-transmitted infections and infectious blindness worldwide.
is a major cause of community-acquired atypical pneumonia.
primarily affects psittacine birds and can be transmitted to humans causing psittacosis, a potentially fatal form of pneumonia. As opposed to other bacterial pathogens, the spread of clinically relevant antimicrobial resistance genes does not seem to be a major problem for the treatment of
infections. However, when exposed to stressing conditions, like those arising from exposure to antimicrobial stimuli, these bacteria undergo a temporary interruption in their replication cycle and enter a viable but non-cultivable state known as persistence. When the stressing conditions are removed,
resumes replication and generation of infectious particles. This review gives an overview of the different survival strategies used by
to evade the deleterious effects of penicillin and IFNγ, with a focus on the different models used to study
persistence, their contribution to elucidating the molecular basis of this complex phenomenon and their potential implications for studies in animal models of infection.
Chlamydia trachomatis, a pathogen responsible for diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to routine molecular ...genetic manipulation. We have developed a combinatorial approach to rapidly generate a comprehensive library of genetically defined mutants. Chemical mutagenesis, coupled with whole-genome sequencing (WGS) and a system for DNA exchange within infected cells, was used to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. As a result, we identified mutants with altered glycogen metabolism, including an attenuated strain defective for type II secretion. The coupling of chemically induced gene variation and WGS to establish genotype–phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.
The obligate intracellular bacterial pathogen Chlamydia trachomatis replicates within a large vacuole or "inclusion" that expands as bacteria multiply but is maintained as an intact organelle. Here, ...we report that the inclusion is encased in a scaffold of host cytoskeletal structures made up of a network of F-actin and intermediate filaments (IF) that act cooperatively to stabilize the pathogen-containing vacuole. Formation of F-actin at the inclusion was dependent on RhoA, and its disruption led to the disassembly of IFs, loss of inclusion integrity, and leakage of inclusion contents into the host cytoplasm. In addition, IF proteins were processed by the secreted chlamydial protease CPAF to form filamentous structures at the inclusion surface with altered structural properties. We propose that Chlamydia has co-opted the function of F-actin and IFs to stabilize the inclusion with a dynamic, structural scaffold while minimizing the exposure of inclusion contents to cytoplasmic innate immune-surveillance pathways.
Chlamydia and Chlamydophila sp. are highly related obligate intracellular bacterial pathogens that cause sexually transmitted diseases, ocular infections and atypical pneumonias. Relatively little is ...known about the molecular mechanisms by which Chlamydiae manipulate the mammalian host because they are intractable to genetic manipulation. Studies with heterologous expression systems have revealed a large set of chlamydial proteins that are potentially translocated into the host cytoplasm (‘effector’ proteins). As new cell biological observations are made and the function of effector proteins begin to be elucidated, a clearer picture of the extent to which Chlamydiae manipulate mammalian cellular processes is beginning to emerge, including the cell cycle, innate immunity, and lipid and membrane transport.
Many intracellular pathogens survive in vacuolar niches composed of host-derived membranes modified extensively by pathogen proteins and lipids. Although intracellular lifestyles offer protection ...from humoral immune responses, vacuole-bound pathogens nevertheless face powerful intracellular innate immune surveillance pathways that can trigger fusion with lysosomes, autophagy, and host cell death. Strategies used by vacuole-bound pathogens to invade and establish a replicative vacuole are well described, but how the integrity and stability of these parasitic vacuoles are maintained is poorly understood. Here, we identify potential mechanisms of pathogenic vacuole maintenance and the consequences of vacuole disruption by highlighting select bacterial and protozoan parasites.
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When Stanley Falkow introduced Molecular Koch’s Postulates (Falkow, 1988) as a conceptual framework to identify microbial factors that contributed to disease, he reaffirmed the ...prominent role that the basic principles of genetic analysis should play in defining genotype-phenotype associations in microbial pathogens. In classical bacterial genetics the nature of mutations is inferred through cis-trans complementation and by indirectly mapping their relative position and physical distance through recombination frequencies — all of which were made possible by the genetic tools of the day: natural transformations, conjugation and transduction. Unfortunately, many of these genetic tools are not always available to study pathogenic bacteria. The recombinant DNA revolution in the 1980s launched the field of molecular pathogenesis as genes could be treated as physical units that could be cut, spliced and transplanted from one microbe to another and thus not only ‘prove’ that an individual gene complemented a virulence defect in a mutant strain but also could impart pathogenic properties to otherwise benign microbes. The recombinant DNA revolution also enabled the generation of newer versions of genetic tools to generate mutations and engineer microbial genomes.
The last decade has ushered in next generation sequencing technologies as a new powerful tool for bacterial genetics. The routine and inexpensive sequencing of microbial genomes has increased the number and phylogenetic scope of microbes that are amenable to functional characterization and experimentation. In this review, we highlight some salient advances in this rapidly evolving area.
Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed 'inclusion') may facilitate ...nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chlamydia trachomatis (Ct) is an intracellular bacterial pathogen that relies on the activity of secreted proteins known as effectors to promote replication and avoidance of immune clearance. ...Understanding the contribution of Ct effectors to pathogenesis has proven to be challenging, given that these proteins often perform multiple functions during intracellular infection. Recent advances in molecular genetic analysis of Ct have provided valuable insights into the multifaceted nature of secreted effector proteins and their impact on the interaction between Ct and host cells and tissues. This review highlights significant findings from genetic analysis of Ct effector functions, shedding light on their diverse roles. We also discuss the challenges faced in this field of study and explore potential opportunities for further research.
•Genetic approaches have significantly accelerated the discovery and characterization of Chlamydia trachomatis effector proteins.•Characterizing Chlamydia effectors is challenging due to their multifunctional nature.•Separation-of-function mutants in Chlamydia effectors are needed to clarify the multiple roles of effectors and their individual contributions to virulence.
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