Genetic diversity in terrestrial subsurface ecosystems impacted by geological degassing

Earth’s mantle releases 38.7 ± 2.9 Tg/yr CO2 along with other reduced and oxidized gases to the atmosphere shaping microbial metabolism at volcanic sites across the globe, yet little is known about its impact on microbial life under non-thermal conditions. Here, we perform comparative metagenomics coupled to geochemical measurements of deep subsurface fluids from a cold-water geyser driven by mantle degassing. Key organisms belonging to uncultivated Candidatus Altiarchaeum show a global biogeographic pattern and site-specific adaptations shaped by gene loss and inter-kingdom horizontal gene transfer. Comparison of the geyser community to 16 other publicly available deep subsurface sites demonstrate a conservation of chemolithoautotrophic metabolism across sites. In silico replication measures suggest a linear relationship of bacterial replication with ecosystems depth with the exception of impacted sites, which show near surface characteristics. Our results suggest that subsurface ecosystems affected by geological degassing are hotspots for microbial life in the deep biosphere.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

The Bovine Tuberculoid Granuloma

The bovine tuberculoid granuloma is the hallmark lesion of bovine tuberculosis (bTB) due to Mycobacterium bovis infection. The pathogenesis of bTB, and thereby the process of bovine tuberculoid granuloma development, involves the recruitment, activation, and maintenance of cells under the influence of antigen, cytokines and chemokines in affected lungs and regional lymph nodes. The granuloma is key to successful control of bTB by preventing pathogen dissemination through containment by cellular and fibrotic layers. Paradoxically, however, it may also provide a niche for bacterial replication. The morphologic and cellular characteristics of granulomas have been used to gauge disease severity in bTB pathogenesis and vaccine efficacy studies. As such, it is critical to understand the complex mechanisms behind granuloma initiation, development, and maintenance.

Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.

Characterizing the Type 6 Secretion System (T6SS) and its role in the virulence of avian pathogenic Escherichia coli strain APECO18

Avian pathogenic E. coli is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes hcp, impK, evpB, vasK and icmF in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal E. coli isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for hcp, impK, evpB, and icmF and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Dictyostelium discoideum. Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of hcp reduced resistance of APECO18 to predation by D. discoideum, suggesting that T6SS is involved in the virulence of APECO18.

Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

Legionella pneumophila, the causative agent of Legionnaires'; disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

Macrophage Selenoproteins Restrict Intracellular Replication of Francisella tularensis and Are Essential for Host Immunity

The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis, a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis-live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo, corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis, mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection.

Pathogen clonal expansion underlies multiorgan dissemination and organ-specific outcomes during murine systemic infection

The dissemination of pathogens through blood and their establishment within organs lead to severe clinical outcomes. However, the within-host dynamics that underly pathogen spread to and clearance from systemic organs remain largely uncharacterized. In animal models of infection, the observed pathogen population results from the combined contributions of bacterial replication, persistence, death, and dissemination, each of which can vary across organs. Quantifying the contribution of each these processes is required to interpret and understand experimental phenotypes. Here, we leveraged STAMPR, a new barcoding framework, to investigate the population dynamics of extraintestinal pathogenic E. coli, a common cause of bacteremia, during murine systemic infection. We show that while bacteria are largely cleared by most organs, organ-specific clearance failures are pervasive and result from dramatic expansions of clones representing less than 0.0001% of the inoculum. Clonal expansion underlies the variability in bacterial burden between animals, and stochastic dissemination of clones profoundly alters the pathogen population structure within organs. Despite variable pathogen expansion events, host bottlenecks are consistent yet highly sensitive to infection variables, including inoculum size and macrophage depletion. We adapted our barcoding methodology to facilitate multiplexed validation of bacterial fitness determinants identified with transposon mutagenesis and confirmed the importance of bacterial hexose metabolism and cell envelope homeostasis pathways for organ-specific pathogen survival. Collectively our findings provide a comprehensive map of the population biology that underlies bacterial systemic infection and a framework for barcode-based high-resolution mapping of infection dynamics.

Spider Mites Singly Infected With Either Wolbachia or Spiroplasma Have Reduced Thermal Tolerance

Heritable symbionts play an essential role in many aspects of host ecology in a temperature-dependent manner. However, how temperature impacts the host and their interaction with endosymbionts remains largely unknown. Here, we investigated the impact of moderate (20°C) and high (30 and 35°C) temperatures on symbioses between the spider mite Tetranychus truncatus and two maternally inherited endosymbionts (Wolbachia and Spiroplasma). We found that the thermal tolerance of mites (as measured by survival after heat exposure) was lower for mites that were singly infected with either Wolbachia or Spiroplasma than it was for co-infected or uninfected mites. Although a relatively high temperature (30°C) is thought to promote bacterial replication, rearing at high temperature (35°C) resulted in losses of Wolbachia and particularly Spiroplasma. Exposing the mites to 20°C reduced the density and transmission of Spiroplasma but not Wolbachia. The four spider mite strains tested differed in the numbers of heat shock genes (Hsps) induced under moderate or high temperature exposure. In thermal preference (Tp) assays, the two Wolbachia-infected spider mite strains preferred a lower temperature than strains without Wolbachia. Our results show that endosymbiont-mediated spider mite responses to temperature stress are complex, involving a combination of changing endosymbiont infection patterns, altered thermoregulatory behavior, and transcription responses.

Transferrins Reduce Replication of Chlamydia suis in McCoy Cells

Chlamydia suis (C. suis) resides in the intestines of pigs and tetracycline-resistant strains are emerging worldwide. Intestinal infections are often subclinical. However, the gut is regarded as a C. suis reservoir and clinical infections have been associated with enteritis, conjunctivitis, pneumonia and reproductive failure. C. suis was found in boar semen and venereal transmission occurred. We studied the anti-Chlamydia suis activity of ovotransferrin (ovoTF) and bovine lactoferrin (bLF). Pre-incubation of C. suis with bLF or ovoTF had no significant effect on overall chlamydia replication (mean fluorescence area) in McCoy cells. The addition of ovoTF to the culture medium had no effect on bacterial replication, but the addition of 0.5 or 5 mg/mL of bLF significantly reduced the inclusion size by 17% and 15% respectively. Egg components are used for cryopreservation of boar semen. When inoculating an ovoTF-containing and Chlamydia suis-spiked semen sample in McCoy cells, a significant reduction in inclusion number (by 7%) and overall replication (by 11%) was observed. Thus, we showed that transferrins possess anti-chlamydial activity. Moreover, ovoTF addition to semen extenders might reduce C. suis venereal transmission. Further research is needed to unravel the mechanisms behind the observations and to enhance the effect of transferrins on C. suis.