Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Comprehensive whole genome sequencing with hybrid assembly of multi-drug resistant Candida albicans isolate causing cerebral abscess.

Comprehensive whole genome sequencing (WGS) with hybrid assembly of a multi-drug resistant (MDR) Candida albicans (CA) isolate causing cerebral abscess was performed using Illumina paired end and Oxford Nanopore long read technologies. The innovative technologies utilized here enabled us to resolve fragmented assemblies, and implement comprehensive and detailed genomic analyses involved in antifungal resistance of Candida spp. Functionally important genes (MDR1, CDR2 and SQN2) involved in antifungal resistance were identified and a phylogenetic analysis of the clinical isolate was performed. Additionally, our clinical isolate was found to share 4 single nucleotide polymorphisms with two other sequenced strains of MDR C. auris (381 and 386) including translation elongation factor EF1α and EF3, ATPase activity associated proteins, and the lysine tRNA ligase.

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae.

Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

Host Cathelicidin Exacerbates Group B Streptococcus Urinary Tract Infection.

Group B Streptococcus (GBS) causes frequent urinary tract infection (UTI) in susceptible populations, including individuals with type 2 diabetes and pregnant women; however, specific host factors responsible for increased GBS susceptibility in these populations are not well characterized. Here, we investigate cathelicidin, a cationic antimicrobial peptide, known to be critical for defense during UTI with uropathogenic Escherichia coli (UPEC). We observed a loss of antimicrobial activity of human and mouse cathelicidins against GBS and UPEC in synthetic urine and no evidence for increased cathelicidin resistance in GBS urinary isolates. Furthermore, we found that GBS degrades cathelicidin in a protease-dependent manner. Surprisingly, in a UTI model, cathelicidin-deficient (Camp-/-) mice showed decreased GBS burdens and mast cell recruitment in the bladder compared to levels in wild-type (WT) mice. Pharmacologic inhibition of mast cells reduced GBS burdens and histamine release in WT but not Camp-/- mice. Streptozotocin-induced diabetic mice had increased bladder cathelicidin production and mast cell recruitment at 24 h postinfection with GBS compared to levels in nondiabetic controls. We propose that cathelicidin is an important immune regulator but ineffective antimicrobial peptide against GBS in urine. Combined, our findings may in part explain the increased frequency of GBS UTI in diabetic and pregnant individuals.IMPORTANCE Certain populations such as diabetic individuals are at increased risk for developing urinary tract infections (UTI), although the underlying reasons for this susceptibility are not fully known. Additionally, diabetics are more likely to become infected with certain types of bacteria, such as group B Streptococcus (GBS). In this study, we find that an antimicrobial peptide called cathelicidin, which is thought to protect the bladder from infection, is ineffective in controlling GBS and alters the type of immune cells that migrate to the bladder during infection. Using a mouse model of diabetes, we observe that diabetic mice are more susceptible to GBS infection even though they also have more infiltrating immune cells and increased production of cathelicidin. Taken together, our findings identify this antimicrobial peptide as a potential contributor to increased susceptibility of diabetic individuals to GBS UTI.

The Fungal Pathogen Candida albicans Promotes Bladder Colonization of Group B Streptococcus.

Group B Streptococcus (GBS) is a common cause of bacterial urinary tract infections (UTI) in susceptible populations, including pregnant women and the elderly. However, the factors that govern GBS persistence and disease severity in this niche are not fully understood. Here, we report that the presence of the fungus Candida albicans, a common urogenital colonizer, can promote GBS UTI. Co-inoculation of GBS with C. albicans increased bacterial adherence to bladder epithelium and promoted GBS colonization in vivo in a C. albicans adhesin-dependent manner. This study demonstrates that fungal colonization of the urogenital tract may be an important determinant of bacterial pathogenesis during UTI.

The murine vaginal microbiota and its perturbation by the human pathogen group B Streptococcus.

BACKGROUND: Composition of the vaginal microbiota has significant influence on female urogenital health and control of infectious disease. Murine models are widely utilized to characterize host-pathogen interactions within the vaginal tract, however, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. Here, we employ 16S rRNA amplicon sequencing to establish the native microbial composition of the vaginal tract in adult C57Bl/6 J mice. We further interrogate the impact of estrous cycle and introduction of the human vaginal pathobiont, group B Streptococcus (GBS) on community state type and stability, and conversely, the impact of the vaginal microbiota on GBS persistence. RESULTS: Sequencing analysis revealed five distinctive community states of the vaginal microbiota dominated largely by Staphylococcus and/or Enterococcus, Lactobacillus, or a mixed population. Stage of estrus did not impact microbial composition. Introduction of GBS decreased community stability at early timepoints; and in some mice, GBS became the dominant bacterium by day 21. Endogenous Staphylococcus abundance correlated with GBS ascension into the uterus, and increased community stability in GBS-challenged mice. CONCLUSIONS: The murine vaginal flora is diverse and fluctuates independently of the estrous cycle. Endogenous flora may impact pathogen colonization and dissemination and should be considered in urogenital infection models.

Tamm-Horsfall Protein Protects the Urinary Tract against Candida albicans.

Urinary tract infections (UTIs) caused by the human fungal pathogen Candida albicans and related species are prevalent in hospitalized patients, especially those on antibiotic therapy, with indwelling catheters, or with predisposing conditions such as diabetes or immunodeficiency. Understanding of key host defenses against Candida UTI is critical for developing effective treatment strategies. Tamm-Horsfall glycoprotein (THP) is the most abundant urine protein, with multiple roles in renal physiology and bladder protection. THP protects against bacterial UTI by blocking bacterial adherence to the bladder epithelium, but its role in defense against fungal pathogens is not yet described. Here we demonstrate that THP restricts colonization of the urinary tract by C. albicans THP binds to C. albicans hyphae, but not the yeast form, in a manner dependent on fungal expression of the Als3 adhesion glycoprotein. THP directly blocks C. albicans adherence to bladder epithelial cells in vitro, and THP-deficient mice display increased fungal burden in a C. albicans UTI model. This work outlines a previously unknown role for THP as an essential component for host immune defense against fungal urinary tract infection.

Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

The Staphylococcus aureus ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst.

Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.

Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection.

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.

Differential Requirements for eIF4E Dose in Normal Development and Cancer.

eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.

MyD88-dependent signaling drives host survival and early cytokine production during Histoplasma capsulatum infection.

The ability of the innate immune system to trigger an adaptive T cell response is critical to resolution of infection with the fungal pathogen Histoplasma capsulatum. However, the signaling pathways and cell types involved in the recognition of and response to this respiratory pathogen remain poorly defined. Here, we show that MyD88, an adaptor protein vital to multiple innate immune pathways, is critically required for the host response to Histoplasma. MyD88-deficient (MyD88-/-) mice are unable to control the fungal burden and are more sensitive to Histoplasma infection than wild-type, Dectin-1-/-, or interleukin 1 receptor-deficient (IL-1R-/-) mice. We found that MyD88 is necessary for the production of key early inflammatory cytokines and the subsequent recruitment of inflammatory monocytes to the lung. In both our in vitro and ex vivo analyses, MyD88 was intrinsically required in dendritic cells and alveolar macrophages for initial cytokine production. Additionally, MyD88-deficient bone marrow-derived dendritic cells fail to efficiently control fungal growth when cocultured with primed splenic T cells. Surprisingly, mice that lack MyD88 only in dendritic cells and alveolar macrophages are competent for early cytokine production and normal survival, indicating the presence of compensatory and redundant MyD88 signaling in other cell types during infection. Ultimately, global MyD88 deficiency prevents proper T cell activation and gamma interferon (IFN-γ) production, which are critical for infection resolution. Collectively, this work reveals a central role for MyD88 in coordinating the innate and adaptive immune responses to infection with this ubiquitous fungal pathogen of humans.

The 3-hydroxy-methylglutaryl coenzyme A lyase HCL1 is required for macrophage colonization by human fungal pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a fungal respiratory pathogen that survives and replicates within the phagolysosome of macrophages. The molecular factors it utilizes to subvert macrophage antimicrobial defenses are largely unknown. Although the ability of H. capsulatum to prevent acidification of the macrophage phagolysosome is thought to be critical for intracellular survival, this hypothesis has not been tested since H. capsulatum mutants that experience decreased phagosomal pH have not been identified. In a screen to identify H. capsulatum genes required for lysis of bone marrow-derived macrophages (BMDMs), we identified an insertion mutation disrupting the H. capsulatum homolog of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) lyase (HCL1). In addition to its inability to lyse macrophages, the hcl1 mutant had a severe growth defect in BMDMs, indicating that HMG CoA lyase gene function is critical for macrophage colonization. In other organisms, HMG CoA lyase catalyzes the last step in the leucine catabolism pathway. In addition, both fungi and humans deficient in HMG CoA lyase accumulate acidic intermediates as a consequence of their inability to catabolize leucine. Consistent with observations in other organisms, the H. capsulatum hcl1 mutant was unable to grow on leucine as the major carbon source, caused acidification of its growth medium in vitro, and resided in an acidified vacuole within macrophages. Mice infected with the hcl1 mutant took significantly longer to succumb to infection than mice infected with the wild-type strain. Taken together, these data indicate the importance of Hcl1 function in H. capsulatum replication in the harsh growth environment of the macrophage phagosome.

Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease.

Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.

Identification of a third msa gene in Renibacterium salmoninarum and the associated virulence phenotype.

Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of < or =10(5) cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10(6) cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.

Expression of duplicate msa genes in the salmonid pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum.