A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Candida-bacterial cross-kingdom interactions.

While the fungus Candida albicans is a common colonizer of healthy humans, it is also responsible for mucosal infections and severe invasive disease. Understanding the mechanisms that allow C. albicans to exist as both a benign commensal and as an invasive pathogen have been the focus of numerous studies, and recent findings indicate an important role for cross-kingdom interactions on C. albicans biology. This review highlights how C. albicans-bacteria interactions influence healthy polymicrobial community structure, host immune responses, microbial pathogenesis, and how dysbiosis may lead to C. albicans infection. Finally, we discuss how cross-kingdom interactions represent an opportunity to identify new antivirulence compounds that target fungal infections.

Intestinal Inflammation Promotes MDL-1+ Osteoclast Precursor Expansion to Trigger Osteoclastogenesis and Bone Loss.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation, but many patients experience extra-intestinal disease. Bone loss is one common extra-intestinal manifestation of IBD that occurs through dysregulated interactions between osteoclasts and osteoblasts. Systemic inflammation has been postulated to contribute to bone loss, but the specific pathologic mechanisms have not yet been fully elucidated. We hypothesized that intestinal inflammation leads to bone loss through increased abundance and altered function of osteoclast progenitors. METHODS: We used chemical, T cell driven, and infectious models of intestinal inflammation to determine the impact of intestinal inflammation on bone volume, the skeletal cytokine environment, and the cellular changes to pre-osteoclast populations within bone marrow. Additionally, we evaluated the potential for monoclonal antibody treatment against an inflammation-induced osteoclast co-receptor, myeloid DNAX activation protein 12-associating lectin-1 (MDL-1) to reduce bone loss during colitis. RESULTS: We observed significant bone loss across all models of intestinal inflammation. Bone loss was associated with an increase in pro-osteoclastogenic cytokines within the bone and an expansion of a specific Cd11b-/loLy6Chi osteoclast precursor (OCP) population. Intestinal inflammation led to altered OCP expression of surface receptors involved in osteoclast differentiation and function, including the pro-osteoclastogenic co-receptor MDL-1. OCPs isolated from mice with intestinal inflammation demonstrated enhanced osteoclast differentiation ex vivo compared to controls, which was abrogated by anti-MDL-1 antibody treatment. Importantly, in vivo anti-MDL-1 antibody treatment ameliorated bone loss during intestinal inflammation. CONCLUSIONS: Collectively, these data implicate the pathologic expansion and altered function of OCPs expressing MDL-1 in bone loss during IBD.

Metabolic Adaptations During Staphylococcus aureus and Candida albicans Co-Infection.

Successful pathogens require metabolic flexibility to adapt to diverse host niches. The presence of co-infecting or commensal microorganisms at a given infection site can further influence the metabolic processes required for a pathogen to cause disease. The Gram-positive bacterium Staphylococcus aureus and the polymorphic fungus Candida albicans are microorganisms that asymptomatically colonize healthy individuals but can also cause superficial infections or severe invasive disease. Due to many shared host niches, S. aureus and C. albicans are frequently co-isolated from mixed fungal-bacterial infections. S. aureus and C. albicans co-infection alters microbial metabolism relative to infection with either organism alone. Metabolic changes during co-infection regulate virulence, such as enhancing toxin production in S. aureus or contributing to morphogenesis and cell wall remodeling in C. albicans. C. albicans and S. aureus also form polymicrobial biofilms, which have greater biomass and reduced susceptibility to antimicrobials relative to mono-microbial biofilms. The S. aureus and C. albicans metabolic programs induced during co-infection impact interactions with host immune cells, resulting in greater microbial survival and immune evasion. Conversely, innate immune cell sensing of S. aureus and C. albicans triggers metabolic changes in the host cells that result in an altered immune response to secondary infections. In this review article, we discuss the metabolic programs that govern host-pathogen interactions during S. aureus and C. albicans co-infection. Understanding C. albicans-S. aureus interactions may highlight more general principles of how polymicrobial interactions, particularly fungal-bacterial interactions, shape the outcome of infectious disease. We focus on how co-infection alters microbial metabolism to enhance virulence and how infection-induced changes to host cell metabolism can impact a secondary infection.

Contemporary Clinical Isolates of Staphylococcus aureus from Pediatric Osteomyelitis Patients Display Unique Characteristics in a Mouse Model of Hematogenous Osteomyelitis.

Osteomyelitis can result from the direct inoculation of pathogens into bone during injury or surgery or from spread via the bloodstream, a condition called hematogenous osteomyelitis (HOM). HOM disproportionally affects children, and more than half of cases are caused by Staphylococcus aureus. Laboratory models of osteomyelitis mostly utilize direct injection of bacteria into the bone or implantation of foreign material and therefore do not directly interrogate the pathogenesis of pediatric hematogenous osteomyelitis. In this study, we inoculated mice intravenously and characterized the resultant musculoskeletal infections using two strains isolated from adults (USA300-LAC and NRS384) and five new methicillin-resistant S. aureus isolates from pediatric osteomyelitis patients. All strains were capable of creating stable infections over 5 weeks, although the incidence varied. Micro-computed tomography (microCT) analysis demonstrated decreases in the trabecular bone volume fraction but little effect on bone cortices. Histological assessment revealed differences in the precise focus of musculoskeletal infection, with various mixtures of bone-centered osteomyelitis and joint-centered septic arthritis. Whole-genome sequencing of three new isolates demonstrated distinct strains, two within the USA300 lineage and one USA100 isolate. Interestingly, this USA100 isolate showed a distinct predilection for septic arthritis compared to the other isolates tested, including NRS384 and LAC, which more frequently led to osteomyelitis or mixed bone and joint infections. Collectively, these data outline the feasibility of using pediatric osteomyelitis clinical isolates to study the pathogenesis of HOM in murine models and lay the groundwork for future studies investigating strain-dependent differences in musculoskeletal infection.

The Yersinia pestis GTPase BipA Promotes Pathogenesis of Primary Pneumonic Plague.

Yersinia pestis is a highly virulent pathogen and the causative agent of bubonic, septicemic, and pneumonic plague. Primary pneumonic plague caused by inhalation of respiratory droplets contaminated with Y. pestis is nearly 100% lethal within 4 to 7 days without antibiotic intervention. Pneumonic plague progresses in two phases, beginning with extensive bacterial replication in the lung with minimal host responsiveness, followed by the abrupt onset of a lethal proinflammatory response. The precise mechanisms by which Y. pestis is able to colonize the lung and survive two very distinct disease phases remain largely unknown. To date, a few bacterial virulence factors, including the Ysc type 3 secretion system, are known to contribute to the pathogenesis of primary pneumonic plague. The bacterial GTPase BipA has been shown to regulate expression of virulence factors in a number of Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhi. However, the role of BipA in Y. pestis has yet to be investigated. Here, we show that BipA is a Y. pestis virulence factor that promotes defense against early neutrophil-mediated bacterial killing in the lung. This work identifies a novel Y. pestis virulence factor and highlights the importance of early bacterial/neutrophil interactions in the lung during primary pneumonic plague.

Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague.

Following inhalation, Yersinia pestis rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in Yersinia spp., the factors mediating early Y. pestis adherence in the lung remain unknown. To identify genes important for Y. pestis adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δcaf1) Y. pestis transposon mutant libraries were serially passaged in vivo to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δcaf1Y. pestis backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for YPO3903 in Y. pestis adherence in the lung and showed that YPO3903 regulated transcript levels of psaA, which encodes a fimbria previously implicated in Y. pestis adherence in vitro Deletion of psaA had a minor effect on Y. pestis adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to psaA By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early Y. pestis adherence during primary pneumonic plague.IMPORTANCE Colonization of the lung by Yersinia pestis is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which Y. pestis adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify Y. pestis genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple Y. pestis adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing Y. pestis adherence and the subsequent development of pneumonic plague.

Inhibition of Neutrophil Primary Granule Release during Yersinia pestis Pulmonary Infection.

Inhalation of Yersinia pestis causes primary pneumonic plague, the most severe manifestation of plague that is characterized by a dramatic neutrophil influx to the lungs. Neutrophils are ineffective during primary pneumonic plague, failing to control Y. pestis growth in the airways. However, the mechanisms by which Y. pestis resists neutrophil killing are incompletely understood. Here, we show that Y. pestis inhibits neutrophil degranulation, an important line of host innate immune defense. We observed that neutrophils from the lungs of mice infected intranasally with Y. pestis fail to release primary granules throughout the course of disease. Using a type III secretion system (T3SS) injection reporter strain, we determined that Y. pestis directly inhibits neutrophil granule release by a T3SS-dependent mechanism. Combinatorial mutant analysis revealed that a Y. pestis strain lacking both effectors YopE and YopH did not inhibit primary granule release and is killed by neutrophils both in vivo and in vitro Similarly, Y. pestis strains injecting only YopE or YopH are able to inhibit the majority of primary granule release from human neutrophils. We determined that YopE and YopH block Rac2 activation and calcium flux, respectively, to inhibit neutrophil primary granule release in isolated human neutrophils. These results demonstrate that Y. pestis coordinates the inhibition of neutrophil primary granule release through the activities of two distinct effectors, and this inhibition promotes Y. pestis survival during primary pneumonic plague.IMPORTANCEYersinia pestis is the causative agent of plague and is one of the deadliest human pathogens. The pneumonic form of Y. pestis infection has played a critical role in the severity of both historical and modern plague outbreaks, yet the host-pathogen interactions that govern the lethality of Yersinia pestis pulmonary infections are incompletely understood. Here, we report that Yersinia pestis inhibits neutrophil degranulation during infection, rendering neutrophils ineffective and allowing unrestricted growth of Y. pestis in the lungs. This coordinated inhibition of granule release not only demonstrates the pathogenic benefit of "silencing" lung neutrophils but also reveals specific host processes and pathways that could be manipulated to reduce the severity of primary pneumonic plague.

Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection.

Neutrophils are the primary immune cell recruited to the site of bacterial infection, where they can rapidly deploy vesicles filled with various pro-inflammatory and anti-microbial proteins. This degranulation process, combined with oxidative and nitrosative mechanisms, is a major part of the initial host response to kill microorganisms. Neutrophils are one of the main cell types that interact with Yersinia pestis during infection, which is often lethal in the absence of prompt antibiotic treatment. Intradermal inoculation of Y. pestis results in bubonic plague, and inhalation of aerosolized droplets containing Y. pestis results in pneumonic plague. Although neutrophils are recruited to the site of inoculation during both bubonic and pneumonic plague, the neutrophils fail to clear Y. pestis, and, during pneumonic plague, contribute to the development of severe pneumonia. Subverting neutrophil responses is critical to the development of fulminant disease, yet the mechanisms by which Y. pestis impairs neutrophils are poorly understood. Cell culture models are important tools for studying Y. pestis interactions with immune cells. We describe a cell culture model for the infection of human neutrophils with Y. pestis. Neutrophils are isolated from human peripheral blood at high purity and subsequently infected with Y. pestis. We specifically focus on the application of this in vitro infection assay to the analysis of neutrophil degranulation responses.

Spatially distinct neutrophil responses within the inflammatory lesions of pneumonic plague.

UNLABELLED: During pneumonic plague, the bacterium Yersinia pestis elicits the development of inflammatory lung lesions that continue to expand throughout infection. This lesion development and persistence are poorly understood. Here, we examine spatially distinct regions of lung lesions using laser capture microdissection and transcriptome sequencing (RNA-seq) analysis to identify transcriptional differences between lesion microenvironments. We show that cellular pathways involved in leukocyte migration and apoptosis are downregulated in the center of lung lesions compared to the periphery. Probing for the bacterial factor(s) important for the alteration in neutrophil survival, we show both in vitro and in vivo that Y. pestis increases neutrophil survival in a manner that is dependent on the type III secretion system effector YopM. This research explores the complexity of spatially distinct host-microbe interactions and emphasizes the importance of cell relevance in assays in order to fully understand Y. pestis virulence. IMPORTANCE: Yersinia pestis is a high-priority pathogen and continues to cause outbreaks worldwide. The ability of Y. pestis to be transmitted via respiratory droplets and its history of weaponization has led to its classification as a select agent most likely to be used as a biological weapon. Unrestricted bacterial growth during the initial preinflammatory phase primes patients to be infectious once disease symptoms begin in the proinflammatory phase, and the rapid disease progression can lead to death before Y. pestis infection can be diagnosed and treated. Using in vivo analyses and focusing on relevant cell types during pneumonic plague infection, we can identify host pathways that may be manipulated to extend the treatment window for pneumonic plague patients.

Yersinia pestis activates both IL-1ß and IL-1 receptor antagonist to modulate lung inflammation during pneumonic plague.

Pneumonic plague is the most rapid and lethal form of Yersinia pestis infection. Increasing evidence suggests that Y. pestis employs multiple levels of innate immune evasion and/or suppression to produce an early "pre-inflammatory" phase of pulmonary infection, after which the disease is highly inflammatory in the lung and 100% fatal. In this study, we show that IL-1ß/IL-18 cytokine activation occurs early after bacteria enter the lung, and this activation eventually contributes to pulmonary inflammation and pathology during the later stages of infection. However, the inflammatory effects of IL-1ß/IL-1-receptor ligation are not observed during this first stage of pneumonic plague. We show that Y. pestis also activates the induction of IL-1 receptor antagonist (IL-1RA), and this activation likely contributes to the ability of Y. pestis to establish the initial pre-inflammatory phase of disease.

Evasion of killing by human antibody and complement through multiple variations in the surface oligosaccharide of Haemophilus influenzae.

The lipopolysaccharide (LPS) of H. influenzae is highly variable. Much of the structural diversity is derived from phase variation, or high frequency on-off switching, of molecules attached during LPS biosynthesis. In this study, we examined the dynamics of LPS phase variation following exposure to human serum as a source of antibody and complement in multiple H. influenzae isolates. We show that lic2A, lgtC and lex2A switch from phase-off to phase-on following serial passage in human serum. These genes, which control attachment of a galα1-4gal di-galactoside structure (lic2A and lgtC phase-on) or an alternative glucose extension (lex2A phase-on) from the same hexose moiety, reduce binding of bactericidal antibody to conserved inner core LPS structures. The effects of the di-galactoside and alternative glucose extension were also examined in the context of the additional LPS phase variable structures phosphorylcholine (ChoP) and sialic acid. We found that di-galactoside, the alternative glucose extension, ChoP, and sialic acid each contribute independently to bacterial survival in the presence of human complement, and have an additive effect in combination. We propose that LPS phase variable extensions serve to shield conserved inner core structures from recognition by host immune components encountered during infection.