Host-derived protease promotes aggregation of Staphylococcus aureus by cleaving the surface protein SasG.

Staphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 µM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

Aging-Associated Augmentation of Gut Microbiome Virulence Capability Drives Sepsis Severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition but also an overabundance of genomic virulence factors that have functional consequence on host immune evasion. IMPORTANCE Older adults suffer more frequent and worse outcomes from sepsis, a critical illness secondary to infection. The reasons underlying this unique susceptibility are incompletely understood. Prior work in this area has focused on how the immune response changes with age. The current study, however, focuses instead on alterations in the community of bacteria that humans live with within their gut (i.e., the gut microbiome). The central concept of this paper is that the bacteria in our gut evolve along with the host and "age," making them more efficient at causing sepsis.

Aging-associated augmentation of gut microbiome virulence capability drives sepsis severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host, but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition, but an overabundance of genomic virulence factors that have functional consequence on host immune evasion. One Sentence Summary: The severity of sepsis in the aged host is in part mediated by longevity-associated increases in gut microbial virulence.

Group B Streptococcus adaptation promotes survival in a hyperinflammatory diabetic wound environment.

Diabetic wounds have poor healing outcomes due to the presence of numerous pathogens and a dysregulated immune response. Group B Streptococcus (GBS) is commonly isolated from diabetic wound infections, but the mechanisms of GBS virulence during these infections have not been investigated. Here, we develop a murine model of GBS diabetic wound infection and, using dual RNA sequencing, demonstrate that GBS infection triggers an inflammatory response. GBS adapts to this hyperinflammatory environment by up-regulating virulence factors including those known to be regulated by the two-component system covRS, such as the surface protein pbsP, and the cyl operon, which is responsible for hemolysin/pigmentation production. We recover hyperpigmented/hemolytic GBS colonies from the murine diabetic wound, which we determined encode mutations in covR. We further demonstrate that GBS mutants in cylE and pbsP are attenuated in the diabetic wound. This foundational study provides insight into the pathogenesis of GBS diabetic wound infections.

Heparan sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE.

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (AgerAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, AgerAHA/AHA mice phenocopied Ager-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of AgerAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Ager-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in AgerAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), AgerAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Ager-/- mice.

Alveolar epithelial glycocalyx degradation mediates surfactant dysfunction and contributes to acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in patients with ARDS. Using mass spectrometry of airspace fluid noninvasively collected from mechanically ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding, which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

Heparin as a therapy for COVID-19: current evidence and future possibilities.

Coronavirus disease 2019 (COVID-19), the clinical syndrome associated with infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted nearly every country in the world. Despite an unprecedented focus of scientific investigation, there is a paucity of evidence-based pharmacotherapies against this disease. Because of this lack of data-driven treatment strategies, broad variations in practice patterns have emerged. Observed hypercoagulability in patients with COVID-19 has created debate within the critical care community on the therapeutic utility of heparin. We seek to provide an overview of the data supporting the therapeutic use of heparin, both unfractionated and low molecular weight, as an anticoagulant for the treatment of SARS-CoV-2 infection. Additionally, we review preclinical evidence establishing biological plausibility for heparin and synthetic heparin-like drugs as therapies for COVID-19 through antiviral and anti-inflammatory effects. Finally, we discuss known adverse effects and theoretical off-target effects that may temper enthusiasm for the adoption of heparin as a therapy in COVID-19 without confirmatory prospective randomized controlled trials. Despite previous failures of anticoagulants in critical illness, plausibility of heparin for COVID-19 is sufficiently robust to justify urgent randomized controlled trials to determine the safety and effectiveness of this therapy.

Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

RATIONALE: Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. OBJECTIVE: To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. METHODS AND RESULTS: The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. CONCLUSION: NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

RNA sequencing analysis of the developing chicken retina.

RNA sequencing transcriptome analysis using massively parallel next generation sequencing technology provides the capability to understand global changes in gene expression throughout a range of tissue samples. Development of the vertebrate retina requires complex temporal orchestration of transcriptional activation and repression. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis. Existing retinal transcriptome projects have been critical to the vision research community for studying aspects of murine and human retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal transcriptome. Here we used Illumina RNA sequencing (RNA-seq) analysis to characterize the mRNA transcriptome of the developing chicken retina in an effort to identify genes critical for retinal development in this important model organism. These data will be valuable to the vision research community for characterizing global changes in gene expression between ocular tissues and critical developmental time points during retinogenesis in the chicken retina.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.