Role of the Yersinia pestis phospholipase D (Ymt) in the initial aggregation step of biofilm formation in the flea.

Transmission of Yersinia pestis by fleas depends on the formation of condensed bacterial aggregates embedded within a gel-like matrix that localizes to the proventricular valve in the flea foregut and interferes with normal blood feeding. This is essentially a bacterial biofilm phenomenon, which at its end stage requires the production of a Y. pestis exopolysaccharide that bridges the bacteria together in a cohesive, dense biofilm that completely blocks the proventriculus. However, bacterial aggregates are evident within an hour after a flea ingests Y. pestis, and the bacterial exopolysaccharide is not required for this process. In this study, we characterized the biochemical composition of the initial aggregates and demonstrated that the yersinia murine toxin (Ymt), a Y. pestis phospholipase D, greatly enhances rapid aggregation following infected mouse blood meals. The matrix of the bacterial aggregates is complex, containing large amounts of protein and lipid (particularly cholesterol) derived from the flea's blood meal. A similar incidence of proventricular aggregation occurred after fleas ingested whole blood or serum containing Y. pestis, and intact, viable bacteria were not required. The initial aggregation of Y. pestis in the flea gut is likely due to a spontaneous physical process termed depletion aggregation that occurs commonly in environments with high concentrations of polymers or other macromolecules and particles such as bacteria. The initial aggregation sets up subsequent binding aggregation mediated by the bacterially produced exopolysaccharide and mature biofilm that results in proventricular blockage and efficient flea-borne transmission. IMPORTANCE: Yersinia pestis, the bacterial agent of plague, is maintained in nature in mammal-flea-mammal transmission cycles. After a flea feeds on a mammal with septicemic plague, the bacteria rapidly coalesce in the flea's digestive tract to form dense aggregates enveloped in a viscous matrix that often localizes to the foregut. This represents the initial stage of biofilm development that potentiates transmission of Y. pestis when the flea later bites a new host. The rapid aggregation likely occurs via a depletion-aggregation mechanism, a non-canonical first step of bacterial biofilm development. We found that the biofilm matrix is largely composed of host blood proteins and lipids, particularly cholesterol, and that the enzymatic activity of a Y. pestis phospholipase D (Ymt) enhances the initial aggregation. Y. pestis transmitted by flea bite is likely associated with this host-derived matrix, which may initially shield the bacteria from recognition by the host's intradermal innate immune response.

Pathogenesis and outcome of VA1 astrovirus infection in the human brain are defined by disruption of neural functions and imbalanced host immune responses.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Cerebral Malaria Is Regulated by Host-Mediated Changes in Plasmodium Gene Expression.

Cerebral malaria (CM), the deadliest complication of Plasmodium infection, is a complex and unpredictable disease. However, our understanding of the host and parasite factors that cause CM is limited. Using a mouse model of CM, experimental CM (ECM), we performed a three-way comparison between ECM-susceptible C57BL/6 mice infected with ECM-causing Plasmodium ANKA parasites [ANKA(C57BL/6)], ECM-resistant BALB/c mice infected with Plasmodium ANKA [ANKA(BALB/c)], and C57BL/6 mice infected with Plasmodium NK65 that does not cause ECM [NK65(C57BL/6)]. All ANKA(C57BL/6) mice developed CM. In contrast, in ANKA(BALB/c) and NK65(C57BL/6), infections do not result in CM and proceed similarly in terms of parasite growth, disease course, and host immune response. However, parasite gene expression in ANKA(BALB/c) was remarkably different than that in ANKA(C57BL/6) but similar to the gene expression in NK65(C57BL/6). Thus, Plasmodium ANKA has an ECM-specific gene expression profile that is activated only in susceptible hosts, providing evidence that the host has a critical influence on the outcome of infection. IMPORTANCE Hundreds of thousands of lives are lost each year due to the brain damage caused by malaria disease. The overwhelming majority of these deaths occur in young children living in sub-Saharan Africa. Thus far, there are no vaccines against this deadly disease, and we still do not know why fatal brain damage occurs in some children while others have milder, self-limiting disease progression. Our research provides an important clue to this problem. Here, we showed that the genetic background of the host has an important role in determining the course and the outcome of the disease. Our research also identified parasite molecules that can potentially be targeted in vaccination and therapy approaches.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

High-Fat High-Sugar Diet-Induced Changes in the Lipid Metabolism Are Associated with Mildly Increased COVID-19 Severity and Delayed Recovery in the Syrian Hamster.

Pre-existing comorbidities such as obesity or metabolic diseases can adversely affect the clinical outcome of COVID-19. Chronic metabolic disorders are globally on the rise and often a consequence of an unhealthy diet, referred to as a Western Diet. For the first time in the Syrian hamster model, we demonstrate the detrimental impact of a continuous high-fat high-sugar diet on COVID-19 outcome. We observed increased weight loss and lung pathology, such as exudate, vasculitis, hemorrhage, fibrin, and edema, delayed viral clearance and functional lung recovery, and prolonged viral shedding. This was accompanied by an altered, but not significantly different, systemic IL-10 and IL-6 profile, as well as a dysregulated serum lipid response dominated by polyunsaturated fatty acid-containing phosphatidylethanolamine, partially recapitulating cytokine and lipid responses associated with severe human COVID-19. Our data support the hamster model for testing restrictive or targeted diets and immunomodulatory therapies to mediate the adverse effects of metabolic disease on COVID-19.

Western diet increases COVID-19 disease severity in the Syrian hamster.

Pre-existing comorbidities such as obesity or metabolic diseases can adversely affect the clinical outcome of COVID-19. Chronic metabolic disorders are globally on the rise and often a consequence of an unhealthy diet, referred to as a Western Diet. For the first time in the Syrian hamster model, we demonstrate the detrimental impact of a continuous high-fat high-sugar diet on COVID-19 outcome. We observed increased weight loss and lung pathology, such as exudate, vasculitis, hemorrhage, fibrin, and edema, delayed viral clearance and functional lung recovery, and prolonged viral shedding. This was accompanied by an increased trend of systemic IL-10 and IL-6, as well as a dysregulated serum lipid response dominated by polyunsaturated fatty acid-containing phosphatidylethanolamine, recapitulating cytokine and lipid responses associated with severe human COVID-19. Our data support the hamster model for testing restrictive or targeted diets and immunomodulatory therapies to mediate the adverse effects of metabolic disease on COVID-19.

Classical complement and inflammasome activation converge in CD14highCD16- monocytes in HIV associated TB-immune reconstitution inflammatory syndrome.

Inflammasome-derived cytokines, IL-1ß and IL-18, and complement cascade have been independently implicated in the pathogenesis of tuberculosis (TB)-immune reconstitution inflammatory syndrome (TB-IRIS), a complication affecting HIV+ individuals starting antiretroviral therapy (ART). Although sublytic deposition of the membrane attack complex (MAC) has been shown to promote NLRP3 inflammasome activation, it is unknown whether these pathways may cooperatively contribute to TB-IRIS. To evaluate the activation of inflammasome, peripheral blood mononuclear cells (PBMCs) from HIV-TB co-infected patients prior to ART and at the IRIS or equivalent timepoint were incubated with a probe used to assess active caspase-1/4/5 followed by screening of ASC (apoptosis-associated speck-like protein containing a CARD domain) specks as a readout of inflammasome activation by imaging flow cytometry. We found higher numbers of monocytes showing spontaneous caspase-1/4/5+ASC-speck formation in TB-IRIS compared to TB non-IRIS patients. Moreover, numbers of caspase-1/4/5+ASC-speck+ monocytes positively correlated with IL-1ß/IL-18 plasma levels. Besides increased systemic levels of C1q and C5a, TB-IRIS patients also showed elevated C1q and C3 deposition on monocyte cell surface, suggesting aberrant classical complement activation. A clustering tSNE analysis revealed TB-IRIS patients are enriched in a CD14highCD16- monocyte population that undergoes MAC deposition and caspase-1/4/5 activation compared to TB non-IRIS patients, suggesting complement-associated inflammasome activation during IRIS events. Accordingly, PBMCs from patients were more sensitive to ex-vivo complement-mediated IL-1ß secretion than healthy control cells in a NLRP3-dependent manner. Therefore, our data suggest complement-associated inflammasome activation may fuel the dysregulated TB-IRIS systemic inflammatory cascade and targeting this pathway may represent a novel therapeutic approach for IRIS or related inflammatory syndromes.

Virus infection of the CNS disrupts the immune-neural-synaptic axis via induction of pleiotropic gene regulation of host responses.

Treatment for many viral infections of the central nervous system (CNS) remains only supportive. Here we address a remaining gap in our knowledge regarding how the CNS and immune systems interact during viral infection. By examining the regulation of the immune and nervous system processes in a nonhuman primate model of West Nile virus neurological disease, we show that virus infection disrupts the homeostasis of the immune-neural-synaptic axis via induction of pleiotropic genes with distinct functions in each component of the axis. This pleiotropic gene regulation suggests an unintended off-target negative impact of virus-induced host immune responses on the neurotransmission, which may be a common feature of various viral infections of the CNS.

A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity.

The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host's ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (TH1)-type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell-specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections.

Vaccine Protection against Multidrug-Resistant Klebsiella pneumoniae in a Nonhuman Primate Model of Severe Lower Respiratory Tract Infection.

Klebsiella pneumoniae is a human gut communal organism and notorious opportunistic pathogen. The relative high burden of asymptomatic colonization by K. pneumoniae is often compounded by multidrug resistance-a potential problem for individuals with significant comorbidities or other risk factors for infection. A carbapenem-resistant K. pneumoniae strain classified as multilocus sequence type 258 (ST258) is widespread in the United States and is usually multidrug resistant. Thus, treatment of ST258 infections is often difficult. Inasmuch as new preventive and/or therapeutic measures are needed for treatment of such infections, we developed an ST258 pneumonia model in cynomolgus macaques and tested the ability of an ST258 capsule polysaccharide type 2 (CPS2) vaccine to moderate disease severity. Compared with sham-vaccinated animals, those vaccinated with ST258 CPS2 had significantly less disease as assessed by radiography 24 h after intrabronchial installation of 108 CFU of ST258. All macaques vaccinated with CPS2 ultimately developed ST258-specific antibodies that significantly enhanced serum bactericidal activity and killing of ST258 by macaque neutrophils ex vivo Consistent with a protective immune response to CPS2, transcripts encoding inflammatory mediators were increased in infected lung tissues obtained from CPS-vaccinated animals compared with control, sham-vaccinated macaques. Taken together, our data provide support for the idea that vaccination with ST258 CPS can be used to prevent or moderate infections caused by ST258. As with studies performed decades earlier, we propose that this prime-boost vaccination approach can be extended to include multiple capsule types.IMPORTANCE Multidrug-resistant bacteria continue to be a major problem worldwide, especially among individuals with significant comorbidities and other risk factors for infection. K. pneumoniae is among the leading causes of health care-associated infections, and the organism is often resistant to multiple classes of antibiotics. A carbapenem-resistant K. pneumoniae strain known as multilocus sequence type 258 (ST258) is the predominant carbapenem-resistant Enterobacteriaceae in the health care setting in the United States. Infections caused by ST258 are often difficult to treat and new prophylactic measures and therapeutic approaches are needed. To that end, we developed a lower respiratory tract infection model in cynomolgus macaques in which to test the ability of ST258 CPS to protect against severe ST258 infection.

Cytokine Diversity in Human Peripheral Blood Eosinophils: Profound Variability of IL-16.

Eosinophilic leukocytes develop in the bone marrow and migrate from peripheral blood to tissues, where they maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. In this study, we explore human eosinophil heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated cytokines varied on a continuum from minimally (coefficient of variation [CV] ≤ 50%) to moderately variable (50% < CV ≤ 90%). Within the moderately variable group, we detected immunoreactive IL-27 (953 ± 504 pg/mg lysate), a mediator not previously associated with human eosinophils. However, our major finding was the distinct and profound variability of eosinophil-associated IL-16 (CV = 103%). Interestingly, eosinophil IL-16 content correlated directly with body mass index (R 2 = 0.60, ***p < 0.0001) in one donor subset. We found no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, lymphocytes, or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized -295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1ß, IL-1α, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration.

Coxiella burnetii RpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth.

Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii ΔrpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii ΔrpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii ΔrpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth.IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.

Differential Gene Expression Patterns of Yersinia pestis and Yersinia pseudotuberculosis during Infection and Biofilm Formation in the Flea Digestive Tract.

Yersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in the Yersinia pseudotuberculosis progenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performed in vivo transcriptional profiling of Y. pestis, a Y. pseudotuberculosis wild-type strain (unable to form biofilm in the flea foregut), and a Y. pseudotuberculosis mutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, the Y. pseudotuberculosis mutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of the hms genes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. The Y. pseudotuberculosis mutant uniquely expressed much higher levels of Yersinia type VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage by Y. pseudotuberculosis but not for blockage by Y. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection. IMPORTANCE Y. pestis emerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes from Y. pseudotuberculosis. Parallel comparisons of the in vitro and in vivo transcriptomes of Y. pestis and two Y. pseudotuberculosis variants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into how Y. pestis has adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

Differential Ability of Pandemic and Seasonal H1N1 Influenza A Viruses To Alter the Function of Human Neutrophils.

Neutrophils are essential cells of host innate immunity. Although the role of neutrophils in defense against bacterial and fungal infections is well characterized, there is a relative paucity of information about their role against viral infections. Influenza A virus (IAV) infection can be associated with secondary bacterial coinfection, and it has long been posited that the ability of IAV to alter normal neutrophil function predisposes individuals to secondary bacterial infections. To better understand this phenomenon, we evaluated the interaction of pandemic or seasonal H1N1 IAV with human neutrophils isolated from healthy persons. These viruses were ingested by human neutrophils and elicited changes in neutrophil gene expression that are consistent with an interferon-mediated immune response. The viability of neutrophils following coculture with either pandemic or seasonal H1N1 IAV was similar for up to 18 h of culture. Notably, neutrophil exposure to seasonal (but not pandemic) IAV primed these leukocytes for enhanced functions, including production of reactive oxygen species and bactericidal activity. Taken together, our results are at variance with the universal idea that IAV impairs neutrophil function directly to predispose individuals to secondary bacterial infections. Rather, we suggest that some strains of IAV prime neutrophils for enhanced bacterial clearance. IMPORTANCE A long-standing notion is that IAV inhibits normal neutrophil function and thereby predisposes individuals to secondary bacterial infections. Here we report that seasonal H1N1 IAV primes human neutrophils for enhanced killing of Staphylococcus aureus. Moreover, we provide a comprehensive view of the changes in neutrophil gene expression during interaction with seasonal or pandemic IAV and report how these changes relate to functions such as bactericidal activity. This study expands our knowledge of IAV interactions with human neutrophils.

Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Mechanism of Gene Regulation by a Staphylococcus aureus Toxin.

The virulence of many bacterial pathogens, including the important human pathogen Staphylococcus aureus, depends on the secretion of frequently large amounts of toxins. Toxin production involves the need for the bacteria to make physiological adjustments for energy conservation. While toxins are primarily targets of gene regulation, such changes may be accomplished by regulatory functions of the toxins themselves. However, mechanisms by which toxins regulate gene expression have remained poorly understood. We show here that the staphylococcal phenol-soluble modulin (PSM) toxins have gene regulatory functions that, in particular, include inducing expression of their own transport system by direct interference with a GntR-type repressor protein. This capacity was most pronounced in PSMs with low cytolytic capacity, demonstrating functional specification among closely related members of that toxin family during evolution. Our study presents a molecular mechanism of gene regulation by a bacterial toxin that adapts bacterial physiology to enhanced toxin production. IMPORTANCE: Toxins play a major role in many bacterial diseases. When toxins are produced during infection, the bacteria need to balance this energy-consuming task with other physiological processes. However, it has remained poorly understood how toxins can impact gene expression to trigger such adaptations. We found that specific members of a toxin family in the major human pathogen Staphylococcus aureus have evolved for gene regulatory purposes. These specific toxins interact with a DNA-binding regulator protein to enable production of the toxin export machinery and ascertain that the machinery is not expressed when toxins are not made and it is not needed. Our study gives mechanistic insight into how toxins may directly adjust bacterial physiology to times of toxin production during infection.

Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells.

UNLABELLED: Tick-borne flaviviruses (TBFVs) cause febrile illnesses, which may progress to severe encephalitis and/or death in humans globally. Most people who recover from severe acute disease suffer from debilitating neurological sequelae, which may be due to viral persistence, infection-induced neurological cell damage, host response, or some combination of these. Acute TBFV infection of human embryonic kidney (HEK) 293T cells in vitro results in the death of >95% of infected cells by day 5. However, replacing cell growth medium allows surviving cells to repopulate and become persistently infected for extended periods of time. The mechanisms responsible for initiation and maintenance of viral persistence remain vague. We subjected the HEK 293T cell transcriptome to deep sequencing to identify genes differentially expressed during acute infection and persistent infection. A total of 451 genes showed unique significant differential expression levels in persistently infected cells relative to the acute phase of infection. Ingenuity Pathway Analysis results suggested that the expression of prosurvival oncogenes AKT2 and ERBB2 was upregulated in persistently infected cells, whereas proapoptotic genes, such as Bad and the beta interferon 1 (IFN-ß1) gene, were downregulated. Genes encoding antiviral cytokines such as the CCL5, tumor necrosis factor alpha (TNF-α), and CXCL10 genes were upregulated during the acute phase, but the same genes were relatively quiescent in persistently infected cells. Exogenous induction of apoptosis demonstrated that persistently infected cells were resistant to apoptosis in a dose-dependent manner. In summary, the differential transcriptome profiles of acute-phase compared to persistently infected HEK 293T cells demonstrated an evasion of apoptosis, which may be critical for a chronic TBFV infection state. These results provide a basis for further study of the mechanisms of TBFV persistence. IMPORTANCE: Tick-borne flaviviruses (TBFVs) cause life-threatening encephalitic disease in humans worldwide. Some people who recover from severe disease may suffer prolonged neurological symptoms due to either virus- or host response-induced cell damage or a combination of the two that are linked to viral persistence. By examining the genes that are significantly differentially expressed in acute TBFV infection versus persistent TBFV infection, we may be able to find the molecular basis of viral persistence. Here we used deep sequencing of the host cell transcriptome to discover that the expression levels of prosurvival genes were upregulated in persistently infected cells relative to acute TBFV infections whereas the expression levels of genes that promote programmed cell death were downregulated. In addition, persistently infected cells were also resistant to exogenous chemical induction of cell death, in a dose-dependent manner, compared to uninfected cells. Our results pave the way for further studies aimed at understanding the precise mechanisms of TBFV persistence.