Cryo-electron tomography of stationary phase Burkholderia thailandensis.

Burkholderia species belonging to the pseudomallei group include significant human and animal pathogens as well as the non-pathogenic species Burkholderia thailandensis . These bacteria co-opt the host cell machinery for their replication and spread between host cells. Thus, it is of interest to understand the structural features of these cells that contribute to host cell colonization and virulence. This study provides high-resolution cryo-electron tomograms of stationary phase Burkholderia thailandensis . It reveals the presence of compact nucleoids and storage granules, as well as examples of the type III secretion system and chemoreceptor arrays. The data can be used to investigate the near-atomic structure of stationary-phase bacterial macromolecules, such as ribosomes.

Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

FAZ assembly in bloodstream form Trypanosoma brucei requires kinesin KIN-E.

Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane via the flagellum attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA missegregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei.

Manipulation of host cell plasma membranes by intracellular bacterial pathogens.

Manipulation of the host cell plasma membrane is critical during infection by intracellular bacterial pathogens, particularly during bacterial entry into and exit from host cells. To manipulate host cells, bacteria deploy secreted proteins that modulate or modify host cell components. Here, we review recent advances that suggest common themes by which bacteria manipulate the host cell plasma membrane. One theme is that bacteria use diverse strategies to target or influence host cell plasma membrane composition and shape. A second theme is that bacteria take advantage of host cell plasma membrane-associated pathways such as signal transduction, endocytosis, and exocytosis. Future investigation into how bacterial and host factors contribute to plasma membrane manipulation by bacterial pathogens will reveal new insights into pathogenesis and fundamental principles of plasma membrane biology.

A glycine-rich PE_PGRS protein governs mycobacterial actin-based motility.

Many key insights into actin regulation have been derived through examining how microbial pathogens intercept the actin cytoskeleton during infection. Mycobacterium marinum, a close relative of the human pathogen Mycobacterium tuberculosis, polymerizes host actin at the bacterial surface to drive intracellular movement and cell-to-cell spread during infection. However, the mycobacterial factor that commandeers actin polymerization has remained elusive. Here, we report the identification and characterization of the M. marinum actin-based motility factor designated mycobacterial intracellular rockets A (MirA), which is a member of the glycine-rich PE_PGRS protein family. MirA contains an amphipathic helix to anchor into the mycobacterial outer membrane and, surprisingly, also the surface of host lipid droplet organelles. MirA directly binds to and activates the host protein N-WASP to stimulate actin polymerization through the Arp2/3 complex, directing both bacterial and lipid droplet actin-based motility. MirA is dissimilar to known N-WASP activating ligands and may represent a new class of microbial and host actin regulator. Additionally, the MirA-N-WASP interaction represents a model to understand how the enigmatic PE_PGRS proteins contribute to mycobacterial pathogenesis.

A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

Plasma membrane protrusions mediate host cell-cell fusion induced by Burkholderia thailandensis.

Cell-cell fusion is important for biological processes including fertilization, development, immunity, and microbial pathogenesis. Bacteria in the pseudomallei group of the Burkholderia species, including B. thailandensis, spread between host cells by inducing cell-cell fusion. Previous work showed that B. thailandensis-induced cell-cell fusion requires intracellular bacterial motility and a bacterial protein secretion apparatus called the type VI secretion system-5 (T6SS-5), including the T6SS-5 protein VgrG5. However, the cellular-level mechanism of and T6SS-5 proteins important for bacteria-induced cell-cell fusion remained incompletely described. Using live-cell imaging, we found bacteria used actin-based motility to push on the host cell plasma membrane to form plasma membrane protrusions that extended into neighboring cells. Then, membrane fusion occurred within membrane protrusions either proximal to the bacterium at the tip or elsewhere within protrusions. Expression of VgrG5 by bacteria within membrane protrusions was required to promote cell-cell fusion. Furthermore, a second predicted T6SS-5 protein, TagD5, was also required for cell-cell fusion. In the absence of VgrG5 or TagD5, bacteria in plasma membrane protrusions were engulfed into neighboring cells. Our results suggest that the T6SS-5 effectors VgrG5 and TagD5 are secreted within membrane protrusions and act locally to promote membrane fusion.

Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis.

Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.

Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.

Lysine methylation shields an intracellular pathogen from ubiquitylation and autophagy.

Many intracellular pathogens avoid detection by their host cells. However, it remains unknown how they avoid being tagged by ubiquitin, an initial step leading to antimicrobial autophagy. Here, we show that the intracellular bacterial pathogen Rickettsia parkeri uses two protein-lysine methyltransferases (PKMTs) to modify outer membrane proteins (OMPs) and prevent their ubiquitylation. Mutants deficient in the PKMTs were avirulent in mice and failed to grow in macrophages because of ubiquitylation and autophagic targeting. Lysine methylation protected the abundant surface protein OmpB from ubiquitin-dependent depletion from the bacterial surface. Analysis of the lysine-methylome revealed that PKMTs modify a subset of OMPs, including OmpB, by methylation at the same sites that are modified by host ubiquitin. These findings show that lysine methylation is an essential determinant of rickettsial pathogenesis that shields bacterial proteins from ubiquitylation to evade autophagic targeting.

Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of the underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosomes may be important for the function of ARIF-1 in systemic virus spread.

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially infected epithelial cells.

Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.

Systematic review of primary total hip arthroplasty using titanium-titanium modular-neck prostheses: the true risk of revision.

AIMS: Modular-neck femoral implants are used to enable more variability in femoral neck version, offset and length. It has been reported that these implants carry a higher rate of revision. The aim of this review was to assess the overall and cause-specific revision rate of titanium-titanium alloy modular-neck implants in primary total hip arthroplasty (THA). METHODS: A systematic review was conducted following PRISMA guidelines and utilising multiple databases. All results were screened for eligibility. Studies published from 2000 onwards, using a current-generation, titanium-titanium, modular-neck implant were included. Overall and cause-specific revision rates were analysed, comparing to fixed-neck prostheses where applicable. RESULTS: 920 studies were screened. After applying exclusion criteria, 23 were assessed in full and 14 included. These consisted of 12 case series and 2 joint registry analyses. 21,841 patients underwent a modular-neck implant with a weighted mean follow-up of 5.7 years, mean age of 62.4 years, and average body mass index (BMI) of 28.4kg/m2. The overall revision rate was 3.95% and 2.98% for modular and fixed-neck prostheses, respectively. For studies with >5 years follow-up the mean revision rate was 3.08%. There was no difference in cause-specific revision rates by implant design. Mean improvement in Harris Hip Score was 41.9. CONCLUSIONS: At medium-term, revision rates for titanium-titanium primary modular-neck THA are acceptable. These prostheses are a sensible management option in patients with considerable anatomical hip deformity not amenable to correction with standard fixed-neck implants. Patients of male gender, high BMI and requiring prostheses with a larger neck, offset or head are at higher risk of implant failure.

Trypanosomes have divergent kinesin-2 proteins that function differentially in flagellum biosynthesis and cell viability.

Trypanosoma brucei, the causative agent of African sleeping sickness, has a flagellum that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 motor proteins. In this study, we investigated the function of the two T. brucei kinesin-2 proteins, TbKin2a and TbKin2b, in bloodstream form trypanosomes. We found that, compared to kinesin-2 proteins across other phyla, TbKin2a and TbKin2b show greater variation in neck, stalk and tail domain sequences. Both kinesins contributed additively to flagellar lengthening. Silencing TbKin2a inhibited cell proliferation, cytokinesis and motility, whereas silencing TbKin2b did not. TbKin2a was localized on the flagellum and colocalized with IFT components near the basal body, consistent with it performing a role in IFT. TbKin2a was also detected on the flagellar attachment zone, a specialized structure that connects the flagellum to the cell body. Our results indicate that kinesin-2 proteins in trypanosomes play conserved roles in flagellar biosynthesis and exhibit a specialized localization, emphasizing the evolutionary flexibility of motor protein function in an organism with a large complement of kinesins.

Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis.

The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1-6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7-11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15-17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.