Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer.

Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.

Pyroptosis in defense against intracellular bacteria.

Pathogenic microbes invade the human body and trigger a host immune response to defend against the infection. In response, host-adapted pathogens employ numerous virulence strategies to overcome host defense mechanisms. As a result, the interaction between the host and pathogen is a dynamic process that shapes the evolution of the host's immune response. Among the immune responses against intracellular bacteria, pyroptosis, a lytic form of cell death, is a crucial mechanism that eliminates replicative niches for intracellular pathogens and modulates the immune system by releasing danger signals. This review focuses on the role of pyroptosis in combating intracellular bacterial infection. We examine the cell type specific roles of pyroptosis in neutrophils and intestinal epithelial cells. We discuss the regulatory mechanisms of pyroptosis, including its modulation by autophagy and interferon-inducible GTPases. Furthermore, we highlight that while host-adapted pathogens can often subvert pyroptosis, environmental microbes are effectively eliminated by pyroptosis.

Pathogenic Rickettsia spp. as emerging models for bacterial biology.

Our understanding of free-living bacterial models like Escherichia coli far outpaces that of obligate intracellular bacteria, which cannot be cultured axenically. All obligate intracellular bacteria are host-associated, and many cause serious human diseases. Their constant exposure to the distinct biochemical niche of the host has driven the evolution of numerous specialized bacteriological and genetic adaptations, as well as innovative molecular mechanisms of infection. Here, we review the history and use of pathogenic Rickettsia species, which cause an array of vector-borne vascular illnesses, as model systems to probe microbial biology. Although many challenges remain in our studies of these organisms, the rich pathogenic and biological diversity of Rickettsia spp. constitutes a unique backdrop to investigate how microbes survive and thrive in host and vector cells. We take a bacterial-focused perspective and highlight emerging insights that relate to new host-pathogen interactions, bacterial physiology, and evolution. The transformation of Rickettsia spp. from pathogens to models demonstrates how recalcitrant microbes may be leveraged in the lab to tap unmined bacterial diversity for new discoveries. Rickettsia spp. hold great promise as model systems not only to understand other obligate intracellular pathogens but also to discover new biology across and beyond bacteria.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Salmonella engages CDC42 effector protein 1 for intracellular invasion.

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.

Chlamydia pneumoniae Upsurge at Tertiary Hospital, Lausanne, Switzerland.

Chlamydia pneumoniae infection cases have usually accounted for <1.5% of community-acquired respiratory tract infections. Currently, Lausanne, Switzerland is experiencing a notable upsurge in cases, with 28 reported within a span of a few months. This upsurge in cases highlights the need for heightened awareness among clinicians.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Nitroreductase-Responsive Photosensitizers for Selective Imaging and Photo-Inactivation of Intracellular Bacteria.

Intracellular Staphylococcus aureus (S. aureus), especially the methicillin resistant staphylococcus aureus (MRSA), are difficult to detect and eradicate due to the protection by the host cells. Antibacterial photodynamic therapy (aPDT) offers promise in treating intracellular bacteria, provided that selective damage to the bacteria ranther than host cells can be realized. According to the different nitroreductase (NTR) levels in mammalian cells and S. aureus, herein NTR-responsive photosensitizers (PSs) (T)CyI-NO2 were designed and synthesized. The emission and 1O2 generation of (T)CyI-NO2 are quenched by the 4-nitrobenzyl group, but can be specifically switched on by bacterial NTR. Therefore, selective imaging and photo-inactivation of intracellular S. aureus and MRSA were achieved. Our findings may pave the way for the development of more efficient and selective aPDT agents to combat intractable intracellular infections.

TAK1 inhibition elicits mitochondrial ROS to block intracellular bacterial colonization.

Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), known as TAK1, is an intracellular signaling intermediate of inflammatory responses. However, a series of mouse Tak1 gene deletion analyses have revealed that ablation of TAK1 does not prevent but rather elicits inflammation, which is accompanied by elevation of reactive oxygen species (ROS). This has been considered a consequence of impaired TAK1-dependent maintenance of tissue integrity. Contrary to this view, here we propose that TAK1 inhibition-induced ROS are an active cellular process that targets intracellular bacteria. Intracellular bacterial effector proteins such as Yersinia's outer membrane protein YopJ are known to inhibit TAK1 to circumvent the inflammatory host responses. We found that such TAK1 inhibition induces mitochondrial-derived ROS, which effectively destroys intracellular bacteria. Two cell death-signaling molecules, caspase 8 and RIPK3, cooperatively participate in TAK1 inhibition-induced ROS and blockade of intracellular bacterial growth. Our results reveal a previously unrecognized host defense mechanism, which is initiated by host recognition of pathogen-induced impairment in a host protein, TAK1, but not directly of pathogens.

Mitigating Lactate-Associated Immunosuppression against Intracellular Bacteria Using Thermoresponsive Nanoparticles for Septic Arthritis Therapy.

Intracellular bacteria are the major contributor to the intractability of septic arthritis, which are sequestered in macrophages to undermine the innate immune response and avoid the antibacterial effect of antibiotics due to the obstruction of the cell membrane. Herein, we report a thermoresponsive nanoparticle, which consists of a phase-change material shell (fatty acids) and an oxygen-producing core (CaO2-vancomycin). Under external thermal stimulation, the shell of the nanoparticle transforms from a solid phase to a liquid phase. Then the CaO2-Vancomycin core is exposed to the surrounding aqueous solution to release vancomycin and generate Ca(OH)2 and oxygen, thereby depleting accumulated lactate to mitigate lactate-associated immunosuppression, stabilizing hypoxia-inducible factor-1α (HIF-1α) to enhance M1-like polarization of macrophages, and increasing reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. This combined effect between the controlled release of antibiotics and enhancement of host innate immunity provides a promising strategy to combat intracellular bacteria for septic arthritis therapy.

Bacterial conversion of a host weapon into a nutritional signal.

Bacteria engulfed by phagocytic cells must resist oxidation damage and adapt to cellular hypoxia, but the mechanisms involved in this process are not completely elucidated. Recent work by Kim et al. in the Journal of Biological Chemistry investigated how the intracellular pathogen Salmonella enterica activates gene expression required to counteract oxidative damage. The authors show that this bacterium utilizes host oxidative molecules to activate regulatory proteins that enhance the production of effector molecules, counteracting the host weapon NADPH oxidase and inducing a protective response.

Virulence Protein Pgp3 Is Insufficient To Mediate Plasmid-Dependent Infectivity of Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of infectious blindness and sexually transmitted bacterial infection globally. C. trachomatis contains a conserved chlamydial plasmid with eight coding sequences. Plasmid-cured Chlamydia strains are attenuated and display reduced infectivity in cell culture and in vivo genital infection of female mice. Mutants that do not express the plasmid-encoded proteins Pgp3, a secreted protein with unknown function, or Pgp4, a putative regulator of pgp3 and other chromosomal loci, display an infectivity defect similar to plasmid-deficient strains. Our objective was to determine the combined and individual contributions of Pgp3 and Pgp4 to this phenotype. Deletion of pgp3 and pgp4 resulted in an infectivity defect detected by competition assay in cell culture and in mice. The pgp3 locus was placed under the control of an anhydrotetracycline-inducible promoter to examine the individual contributions of Pgp3 and Pgp4 to infectivity. Expression of pgp3 was induced 100- to 1,000-fold after anhydrotetracycline administration, regardless of the presence or absence of pgp4. However, secreted Pgp3 was not detected when pgp4 was deleted, confirming a role for Pgp4 in Pgp3 secretion. We discovered that expression of pgp3 or pgp4 alone was insufficient to restore normal infectivity, which required expression of both Pgp3 and Pgp4. These results suggest Pgp3 and Pgp4 are both required for infectivity during C. trachomatis infection. Future studies are required to determine the mechanism by which Pgp3 and Pgp4 influence chlamydial infectivity as well as the potential roles of Pgp4-regulated loci.

Eat or Be Eaten: Strategies Used by Legionella to Acquire Host-Derived Nutrients and Evade Lysosomal Degradation.

To replicate within host cells, bacterial pathogens must acquire host-derived nutrients while avoiding degradative antimicrobial pathways. Fundamental insights into bacterial pathogenicity have been revealed by bacteria of the genus Legionella, which naturally parasitize free-living protozoa by establishing a membrane-bound replicative niche termed the Legionella-containing vacuole (LCV). Biogenesis of the LCV and intracellular replication rely on rapid evasion of the endocytic pathway and acquisition of host-derived nutrients, much of which is mediated by bacterial effector proteins translocated into host cells by a Dot/Icm type IV secretion system. Billions of years of co-evolution with eukaryotic hosts and broad host tropism have resulted in expansion of the Legionella genome to accommodate a massive repertoire of effector proteins that promote LCV biogenesis, safeguard the LCV from endolysosomal maturation, and mediate the acquisition of host nutrients. This minireview is focused on the mechanisms by which an ancient intracellular pathogen leverages effector proteins and hijacks host cell biology to obtain essential host-derived nutrients and prevent lysosomal degradation.

A potential role for chlamydial infection in rheumatoid arthritis development.

OBJECTIVES: To assess the relationship between self-reported and serologic evidence of prior chlamydial infection, rheumatoid arthritis (RA)-related autoantibodies and risk of RA-development. METHODS: This is a nested study within a prospective Swiss-based cohort including all first-degree relatives of RA patients (RA-FDR) who answered a question on past chlamydial infections. Primary outcome was systemic autoimmunity associated with RA (RA-autoimmunity) defined as positivity for anti-citrullinated peptide antibodies (ACPA) and/or rheumatoid factor (RF). Secondary outcomes were high levels of RA-autoimmunity, RA-associated symptoms and RA-autoimmunity, and subsequent seropositive RA diagnosis. We conducted a nested case-control analysis by measuring the serological status against Chlamydia trachomatis' major outer membrane protein. We replicated our analysis in an independent United States-based RA-FDR cohort. RESULTS: Among 1231 RA-FDRs, 168 (13.6%) developed RA-autoimmunity. Prevalence of self-reported chlamydial infection was significantly higher in individuals with RA-autoimmunity compared with controls (17.9% vs 9.8%, OR = 2.00, 95%CI: 1.27-3.09, p < 0.01). This association remained significant after adjustments (OR = 1.91, 95%CI: 1.20-2.95). Stronger effect sizes were observed in later stages of RA development. There was a similar trend between a positive C. trachomatis serology and high levels of RA-autoimmunity (OR = 3.05, 95% CI: 1.10-8.46, p= 0.032). In the replication cohort, there were significant associations between chlamydial infection and RF positivity and incident RA, but not anti-CCP positivity. CONCLUSIONS: Self-reported chlamydial infections are associated with elevated RA-autoimmunity in at risk individuals. The differing association of chlamydial infections and ACPA/RF between cohorts will need to be explored in future studies but is consistent with a role of mucosal origin of RA-related autoimmunity.

Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Molecular causes of an evolutionary shift along the parasitism-mutualism continuum in a bacterial symbiont.

Symbiosis with microbes is a ubiquitous phenomenon with a massive impact on all living organisms, shaping the world around us today. Theoretical and experimental studies show that vertical transmission of symbionts leads to the evolution of mutualistic traits, whereas horizontal transmission facilitates the emergence of parasitic features. However, these studies focused on phenotypic data, and we know little about underlying molecular changes at the genomic level. Here, we combined an experimental evolution approach with infection assays, genome resequencing, and global gene expression analysis to study the effect of transmission mode on an obligate intracellular bacterial symbiont. We show that a dramatic shift in the frequency of genetic variants, coupled with major changes in gene expression, allow the symbiont to alter its position in the parasitism-mutualism continuum depending on the mode of between-host transmission. We found that increased parasitism in horizontally transmitted chlamydiae residing in amoebae was a result of processes occurring at the infectious stage of the symbiont's developmental cycle. Specifically, genes involved in energy production required for extracellular survival and the type III secretion system-the symbiont's primary virulence mechanism-were significantly up-regulated. Our results identify the genomic and transcriptional dynamics sufficient to favor parasitic or mutualistic strategies.

Rapid Changes to Endomembrane System of Infected Root Nodule Cells to Adapt to Unusual Lifestyle.

Symbiosis between leguminous plants and soil bacteria rhizobia is a refined type of plant-microbial interaction that has a great importance to the global balance of nitrogen. The reduction of atmospheric nitrogen takes place in infected cells of a root nodule that serves as a temporary shelter for thousands of living bacteria, which, per se, is an unusual state of a eukaryotic cell. One of the most striking features of an infected cell is the drastic changes in the endomembrane system that occur after the entrance of bacteria to the host cell symplast. Mechanisms for maintaining intracellular bacterial colony represent an important part of symbiosis that have still not been sufficiently clarified. This review focuses on the changes that occur in an endomembrane system of infected cells and on the putative mechanisms of infected cell adaptation to its unusual lifestyle.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

Autophagy in intracellular bacterial infection.

Autophagy is a conserved intracellular degradation process enclosing the bulk of cytosolic components for lysosomal degradation to maintain cellular homeostasis. Accumulating evidences showed that a specialized form of autophagy, known as xenophagy, could serve as an innate immune response to defend against pathogens invading inside the host cells. Correspondingly, infectious pathogens have developed a variety of strategies to disarm xenophagy, leading to a prolonged and persistent intracellular colonization. In this review, we first summarize the current knowledge about the general mechanisms of intracellular bacterial infections and xenophagy. We then focus on the ongoing battle between these two processes.