scholarly journals Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Dominik Brokatzky ◽  
Oliver Kretz ◽  
Georg Häcker
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Intracellular Bacteria ◽  
Mitochondrial Apoptosis ◽  
Content Type ◽  
Obligate Intracellular ◽  
Human Epithelial Cells ◽  
Antiapoptotic Activity ◽  
Obligate Intracellular Bacteria ◽  
Induced Apoptosis

ABSTRACT Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-XL or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis. Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.

scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Y. Chung ◽  
Amanda M. Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Clp Protease ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Developmental Form ◽  
Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals Stenotrophomonas maltophiliaEncodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, IncludingPseudomonas aeruginosa

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Megan Y. Nas ◽  
Richard C. White ◽  
Ashley L. DuMont ◽  
Alberto E. Lopez ◽  
Nicholas P. Cianciotto
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Secretion System ◽  
Lung Epithelial Cells ◽  
Type Ii Secretion ◽  
Bacterial Cells ◽  
Content Type ◽  
Type Iv ◽  
Lung Epithelial ◽  
Induced Apoptosis

ABSTRACTStenotrophomonas maltophiliais an emerging opportunistic and nosocomial pathogen.S. maltophiliais also a risk factor for lung exacerbations in cystic fibrosis patients.S. maltophiliaattaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed thatS. maltophiliaalso encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved amongS. maltophiliastrains and, looking beyond theStenotrophomonasgenus, is most similar to the T4SS ofXanthomonas. To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains ofPseudomonas aeruginosa, the T4SS promoted the growth ofS. maltophiliaand reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS requiredS. maltophiliacontact with its target. Thus,S. maltophiliaVirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before.

scholarly journals A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicolas Jacquier ◽  
Akhilesh K. Yadav ◽  
Trestan Pillonel ◽  
Patrick H. Viollier ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Lytic Transglycosylase ◽  
Obligate Intracellular Bacteria

ABSTRACT Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. IMPORTANCE Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

scholarly journals New Frontiers in Type III Secretion Biology: the Chlamydia Perspective

2013 ◽  
Vol 82 (1) ◽  
pp. 2-9 ◽  
Author(s):  
K. E. Mueller ◽  
G. V. Plano ◽  
K. A. Fields
Keyword(s):  
Type Iii Secretion ◽  
Genetic Manipulation ◽  
Respiratory Pathogen ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Type Iii ◽  
Obligate Intracellular Bacteria ◽  
Coding Capacity

ABSTRACTMembers of the orderChlamydialescomprise a group of exquisitely evolved parasites of eukaryotic hosts that extends from single-celled amoeba to mammals. The most notable are human pathogens and include the agent of oculogenital diseaseChlamydia trachomatis, the respiratory pathogenC. pneumoniae, and the zoonotic agentC. psittaci. All of these species are obligate intracellular bacteria that develop within parasitophorous vesicles termed inclusions. This demanding lifestyle necessitates orchestrated entry into nonphagocytic cells, creation of a privileged intracellular niche, and subversion of potent host defenses. All chlamydial genomes contain the coding capacity for a nonflagellar type III secretion system, and this mechanism has arisen as an essential contributor to chlamydial virulence. The emergence of tractable approaches to the genetic manipulation of chlamydiae raises the possibility of explosive progress in understanding this important contributor to chlamydial pathogenesis. This minireview considers challenges and recent advances that have revealed how chlamydiae have maintained conserved aspects of T3S while exploiting diversification to yield a system that exerts a fundamental role in the unique biology ofChlamydiaspecies.

scholarly journals Broad Degradation of Proapoptotic Proteins with the Conserved Bcl-2 Homology Domain 3 during Infection with Chlamydia trachomatis

2005 ◽  
Vol 73 (3) ◽  
pp. 1399-1403 ◽  
Author(s):  
Songmin Ying ◽  
Barbara M. Seiffert ◽  
Georg Häcker ◽  
Silke F. Fischer
Keyword(s):  
Chlamydia Trachomatis ◽  
Chlamydial Infection ◽  
Death Receptor ◽  
Proteolytic Degradation ◽  
Intracellular Bacteria ◽  
Obligate Intracellular ◽  
Infected Cells ◽  
Domain 3 ◽  
Obligate Intracellular Bacteria ◽  
Induced Apoptosis

ABSTRACT Chlamydiae are obligate intracellular bacteria that can inhibit apoptosis of their host cell. As shown recently, this inhibition is in part explained by the proteolytic degradation of the proapoptotic Bcl-2 family members (BH3-only proteins) Bim, Puma, and Bad upon chlamydial infection. In this study, we further explore this antiapoptotic mechanism. In cells infected with a Chlamydia trachomatis L2 strain, Bim, Puma, and Bad were degraded with similar kinetics, and the degradation of all three was blocked by inhibition of the proteasome. Furthermore, the BH3-only proteins Bmf, Noxa, and tBid were also targeted by chlamydial infection. The constitutively expressed Bmf disappeared during infection. When Noxa was experimentally induced, the levels were also reduced by infection with C. trachomatis. In death-receptor-induced apoptosis, cleaved and activated tBid was degraded, and this destruction was also prevented by inhibition of the proteasome. These results show that chlamydial infection leads to a broad degradation of BH3-only proteins. This loss of proapoptotic factors can explain the almost general protection of infected cells against apoptotic stimuli.

scholarly journals Azithromycin Kills Invasive Aggregatibacter actinomycetemcomitans in Gingival Epithelial Cells

2012 ◽  
Vol 57 (3) ◽  
pp. 1347-1351 ◽  
Author(s):  
Pin-Chuang Lai ◽  
John D. Walters
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Periodontal Pocket ◽  
Organic Cations ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Gingival Epithelial Cells ◽  
Cell Incubation ◽  
Human Gingival Epithelial Cells

ABSTRACTAggregatibacter actinomycetemcomitansinvades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelialA. actinomycetemcomitans. [3H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers withA. actinomycetemcomitans(Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited byl-carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of bothA. actinomycetemcomitansstrains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (P< 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P< 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelialA. actinomycetemcomitansand is shared with organic cations.

scholarly journals A Coming of Age Story: Chlamydia in the Post-Genetic Era

2015 ◽  
Vol 84 (3) ◽  
pp. 612-621 ◽  
Author(s):  
Anna J. Hooppaw ◽  
Derek J. Fisher
Keyword(s):  
Global Economy ◽  
Financial Burden ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Reticulate Body ◽  
Technological Advances ◽  
Obligate Intracellular Bacteria

Chlamydiaspp. are ubiquitous, obligate, intracellular Gram-negative bacterial pathogens that undergo a unique biphasic developmental cycle transitioning between the infectious, extracellular elementary body and the replicative, intracellular reticulate body. The primaryChlamydiaspecies associated with human disease areC. trachomatis, which is the leading cause of both reportable bacterial sexually transmitted infections and preventable blindness, andC. pneumoniae, which infects the respiratory tract and is associated with cardiovascular disease. Collectively, these pathogens are a significant source of morbidity and pose a substantial financial burden on the global economy. Past efforts to elucidate virulence mechanisms of these unique and important pathogens were largely hindered by an absence of genetic methods. Watershed studies in 2011 and 2012 demonstrated that forward and reverse genetic approaches were feasible withChlamydiaand that shuttle vectors could be selected and maintained within the bacterium. While these breakthroughs have led to a steady expansion of the chlamydial genetic tool kit, there are still roads left to be traveled. This minireview provides a synopsis of the currently available genetic methods forChlamydiaalong with a comparison to the methods used in other obligate intracellular bacteria. Limitations and advantages of these techniques will be discussed with an eye toward the methods still needed, and how the current state of the art for genetics in obligate intracellular bacteria could direct future technological advances forChlamydia.

scholarly journals The growing repertoire of genetic tools for dissecting chlamydial pathogenesis

2021 ◽  
Author(s):  
Arkaprabha Banerjee ◽  
David E Nelson
Keyword(s):  
Genetic Engineering ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Pathogenic Potential ◽  
Host Preferences ◽  
Genetic Tools ◽  
Obligate Intracellular ◽  
Obligate Intracellular Bacteria ◽  
Multiple Species

Abstract Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last ten years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

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