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Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation. Author summary: Respiratory tract infections are highly debilitating, and Haemophilus influenzae is a bacterial pathogen that is associated with persistent acute and chronic respiratory tract infections particularly in vulnerable parts of the population. Persistent infections rely on close molecular interactions between the human respiratory cells and the bacterial pathogen, and here we have investigated changes in host and bacterial cells during persistent, long-term infections with H. influenzae. We were able to show for the first time that H. influenzae infections can induce a tolerance to the presence of bacteria in human respiratory epithelia. This tolerance included reduced immune responses, and required live bacteria to be established, which indicates that H. influenzae likely produce specific effector molecules that interrupt immune system signalling. This is the first time that such interactions have been documented for H. influenzae, and suggests that future treatments for H. influenzae infections could include those that strengthen the human immune responses. [ABSTRACT FROM AUTHOR]

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group. [ABSTRACT FROM AUTHOR]

UnlabelledInhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism.Key messageHMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant human hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the human bactericidal enzyme hGIIA to exert its bactericidal function. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is an exclusively human Gram-positive bacterial pathogen ranked among the 'top 10' causes of infection-related deaths worldwide. GAS commonly causes benign and self-limiting epithelial infections (pharyngitis and impetigo), and less frequent severe invasive diseases (bacteremia, toxic shock syndrome and necrotizing fasciitis). Annually, GAS causes 700 million infections, including 1.8 million invasive infections with a mortality rate of 25%. In order to establish an infection, GAS must counteract the oxidative stress conditions generated by the release of reactive oxygen species (ROS) at the infection site by host immune cells such as neutrophils and monocytes. ROS are the highly reactive and toxic byproducts of oxygen metabolism, including hydrogen peroxide (H2O2), superoxide anion (O2 •-), hydroxyl radicals (OH •) and singlet oxygen (O2*), which can damage bacterial nucleic acids, proteins and cell membranes. This review summarizes the enzymatic and regulatory mechanisms utilized by GAS to thwart ROS and survive under conditions of oxidative stress. [ABSTRACT FROM AUTHOR]

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H 2 O 2 ) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H 2 O 2 exposure, cell integrity decreased ( p <0.05), GSH/GSSG ratio reduced ( p <0.05), and ATP production declined by 83% ( p <0.05) in the sensitive and 55% ( p <0.05) in the resistant group; mtROS production increased 3.4-fold ( p <0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function., Competing Interests: Conflicts of Interest The authors declare no competing interests.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a human pathogen which causes significant morbidity and mortality globally. GAS typically infects the throat and skin of the host, causing mild infections such as pharyngitis and impetigo, in addition to life threatening conditions including necrotizing fasciitis, streptococcal toxic shock syndrome (STSS), and bacteremia. Repeated infection with GAS may result in the non-suppurative sequelae, acute rheumatic fever, and acute glomerulonephritis. GAS remains sensitive to the antibiotic penicillin which can be administered as a means to treat infection or as prophylaxis. However, issues with patient compliance and a growing concern over the possible emergence of resistant GAS strains may limit the usefulness of antibiotics in the future. A vaccine capable of preventing GAS infection may be the only effective way to control and eliminate GAS infection and disease.