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.

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.

Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence.

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of the bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, thereby revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol.

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin-based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin-based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time-lapse microscopy using green fluorescent protein-LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin-based motility moved away from LC3-positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin-based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.

N-Acylated Derivatives of Sulfamethoxazole Block Chlamydia Fatty Acid Synthesis and Interact with FabF.

The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.

Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis Infectivity.

The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 ≤ 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 µM. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.

Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication.

Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C. trachomatis replication. When bacterial replication was inhibited using KSK120, an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C. trachomatis inclusion expansion.

Maladjusted host immune responses induce experimental cerebral malaria-like pathology in a murine Borrelia and Plasmodium co-infection model.

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1ß and TNF-α, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.

Chlamydia pneumoniae infection results in generalized bone loss in mice.

Osteoporosis is associated with a general bone loss. Whether infections could contribute to osteoporosis is not known. Chlamydia pneumoniae causes chronic infections and produces potentially bone resorptive cytokines. The effect of C. pneumoniae infection was investigated in vivo in 10-week old mice (c57BL/6) and in vitro in the human osteoblast-like cell line hFOB 1.19 (hFOB). Bone mineral density (BMD) was measured before and 16 days after infection. C. pneumoniae-infected mice had decreased (p<0.05) total and subcortical BMD at the distal femur and proximal tibia compared with controls, but no body-weight gain differences. IL-6 (56 vs. 39pg/mL, p=0.02) and IL-1beta (11 vs. 0pg/mL, p=0.003) levels in sera, and CD3(+) T-cells (p=0.04) were higher in infected mice compared with controls. In vitro, hFOB infected with C. pneumoniae was associated with increased IL-6 (p=0.01) and RANKL (p<0.05) mRNA expression; additionally, IL-6 secretion increased in a dose-dependent manner (p<0.05). In summary, mice infected with C. pneumoniae had generalized bone loss associated with increased IL-6 and IL-1. In addition, C. pneumoniae established an infection in an osteoblast cell line in vitro with similar cytokine profiles as those in vivo, supporting a causal linkage.

The intrinsic immunoglobulin g endopeptidase activity of streptococcal Mac-2 proteins implies a unique role for the enzymatically impaired Mac-2 protein of M28 serotype strains.

IdeS, a secreted cysteine protease of the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G (IgG). Two allelic variants of the enzyme have been described, the IgG-specific endopeptidase, IdeS (or Mac-1) and Mac-2, a protein with only weak IgG endopeptidase activity, which has been suggested to interfere with opsonophagocytosis by blocking Fcgamma receptors of phagocytic cells. However, despite the fact that Mac-2 proteins interact with Fcgamma receptors, no inhibition of reactive oxygen species (ROS) production, opsonophagocytosis, or streptococcal killing by Mac-2 has been reported. In the present study, Mac-2 proteins are shown to contain IgG endopeptidase activity indistinguishable from the enzymatic activity exhibited by IdeS/Mac-1 proteins. The earlier reported weak IgG endopeptidase activity appears to be unique to Mac-2 of M28 serotype strains (Mac-2(M28)) and is most likely due to the formation of a disulfide bond between the catalytic site cysteine and a cysteine residue in position 257 of Mac-2(M28). Furthermore, Mac-2 proteins are shown to inhibit ROS production ex vivo, independently of the IgG endopeptidase activity of the proteins. Inhibition of ROS generation per se, however, was not sufficient to mediate streptococcal survival in bactericidal assays. Thus, in contrast to earlier studies, implicating separate functions for IdeS and Mac-2 protein variants, the current study suggests that Mac-2 and IdeS are bifunctional proteins, combining Fcgamma receptor binding and IgG endopeptidase activity. This finding implies a unique role for Mac-2 proteins of the M28 serotype, since this serotype has evolved and retained a Mac-2 protein lacking IgG endopeptidase activity.