RESUMO
Pathogen diversity within an infected organism has traditionally been explored through the lens of genetic heterogeneity. Hallmark studies have characterized how genetic diversity within pathogen subpopulations contributes to treatment escape and infectious disease progression. However, recent studies have begun to reveal the mechanisms by which phenotypic heterogeneity is established within genetically identical populations of invading pathogens. Furthermore, exciting new work highlights how these phenotypically heterogeneous subpopulations contribute to a pathogen population better equipped to handle the complex and fluctuating environment of a host organism. In this review, we focus on how bacterial pathogens, including Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, establish and maintain phenotypic heterogeneity, and we explore recent work demonstrating causative links between this heterogeneity and infection outcome.
RESUMO
Antibiotic-resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs but do not inhibit bacterial growth. Here, we describe the identification of an isomer, 4EpDN, that is 2-fold more potent (50% inhibitory concentration [IC50] of 4 µM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injectisome T3SS. 4EpDN reduced the number of T3SS needles detected on the surface of Yersinia pseudotuberculosis as detected by microscopy. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.
Assuntos
Sistemas de Secreção Tipo III , Yersinia pseudotuberculosis , Proteínas de Bactérias/genética , Células HeLa , Humanos , Pseudomonas aeruginosa , Sistemas de Secreção Tipo III/genética , Virulência , Yersinia pseudotuberculosis/genéticaRESUMO
Closely related bacterial genomes usually differ in gene content, suggesting that nearly every strain in nature may be ecologically unique. We have tested this hypothesis by sequencing the genomes of extremely close relatives within a recognized taxon and analyzing the genomes for evidence of ecological distinctness. We compared the genomes of four Death Valley isolates plus the laboratory strain W23, all previously classified as Bacillus subtilis subsp. spizizenii and hypothesized through multilocus analysis to be members of the same ecotype (an ecologically homogeneous population), named putative ecotype 15 (PE15). These strains showed a history of positive selection on amino acid sequences in 38 genes. Each of the strains was under a different regimen of positive selection, suggesting that each strain is ecologically unique and represents a distinct ecological speciation event. The rate of speciation appears to be much faster than can be resolved with multilocus sequencing. Each PE15 strain contained unique genes known to confer a function for bacteria. Remarkably, no unique gene conferred a metabolic system or subsystem function that was not already present in all the PE15 strains sampled. Thus, the origin of ecotypes within this clade shows no evidence of qualitative divergence in the set of resources utilized. Ecotype formation within this clade is consistent with the nanoniche model of bacterial speciation, in which ecotypes use the same set of resources but in different proportions, and genetic cohesion extends beyond a single ecotype to the set of ecotypes utilizing the same resources.
Assuntos
Bacillus subtilis/genética , Ecossistema , Genoma Bacteriano , Bacillus subtilis/classificação , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Genômica , Dados de Sequência Molecular , Filogenia , Seleção GenéticaRESUMO
Chlamydia trachomatis, a leading cause of bacteria sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs) into the inclusion membrane. Here, we characterize IncE, a multi-functional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C-terminus. The proximal SLiM mimics an R-SNARE motif to recruit syntaxin (STX) 7 and 12-containing vesicles to the inclusion. The distal SLiM mimics the Sorting Nexin (SNX) 5 and 6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding to two distinct vesicle classes, IncE reprograms host cell trafficking to promote the formation of a class of hybrid vesicles at the inclusion that are required for C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to facilitate rapid evolution in a limited protein space to disrupt host cell processes.
RESUMO
Chlamydia trachomatis, a leading cause of bacterial sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs [inclusion membrane proteins]) into the inclusion membrane. Here, we characterize IncE, a multifunctional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C terminus. The proximal SLiM, by mimicking just a small portion of an R-N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) motif, binds and recruits syntaxin (STX)7- and STX12-containing vesicles to the inclusion. The distal SLiM mimics the sorting nexin (SNX)5 and SNX6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding two distinct vesicle classes, IncE brings these vesicles in close apposition with each other at the inclusion to facilitate C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to enable rapid evolution in a limited protein space to disrupt host cell processes.
Assuntos
Proteínas de Bactérias , Chlamydia trachomatis , Chlamydia trachomatis/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Células HeLa , Motivos de Aminoácidos , Transporte Proteico , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Proteínas Qa-SNARE/metabolismo , Ligação ProteicaRESUMO
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inc lusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen activated protein kinase kinase kinase 2 (MEKK2) and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection. Importance: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrate that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections, but also in understanding the role of TRAF7 in cancer.
RESUMO
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.
Assuntos
Proteínas de Bactérias , Infecções por Chlamydia , Chlamydia trachomatis , Interações Hospedeiro-Patógeno , Humanos , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/imunologia , Células HeLa , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/imunologia , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Imunidade Inata , Ligação Proteica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células HEK293RESUMO
Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartmentthe inclusionand secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.