Biology subjects

Leong, J. M.

Publications and source records attributed to Leong, J. M..

15 recordsLinked to original sources

bioRxiv2025

Retrospective analysis of Pseudomonas aeruginosa clinical isolates for secreted PrpL protease: a key virulence factor associated with corneal tissue damage

BackgroundPseudomonas aeruginosa is a ubiquitous organism that adapts well in different environments. It is an opportunistic bacterial pathogen that produces a wide range of virulence factors, colonizes lungs to cause pneumonia, causes non-healing wounds especially in burn victims, and is a major culprit in destructive keratitis. It can reach the cornea through reusable, extended-use contact lenses and by contaminated eyedrops and artificial tears. Secreted proteases of P. aeruginosa together with pyocyanin metabolite, which inhibits Serine Protease Inhibitors (Serpins) activity contribute to severe tissue damage during infection. MethodsP. aeruginosa strains isolated from different clinical sites were obtained from different researchers and clinicians for this study. We examined P. aeruginosa strains and secretion defective and prpL knockout mutants in PA64481 strain for lysyl endopeptidase (PrpL, a serine protease that cleaves after a lysine residue) activity using serine protease specific D-Val-Leu-Lys-p-nitroanalide substrate. We also determined pyocyanin production in these strains. ResultsExamination of secreted milieu from P. aeruginosa showed that 41 corneal isolates had detectable lysyl endopeptidase activity associated with PrpL at levels significantly higher than by 27 non-corneal isolates. We found that PrpL is secreted by the xcp-based type II secretion system. Bacterial culture supernatants displaying higher PrpL activity and not low secreted PrpL levels disrupted corneal epithelial cell monolayers in vitro, which is consistent with a role of this protease in destructive keratitis. Many examined strains also produced high levels of pyocyanin. ConclusionsThis retrospective examination of clinical P. aeruginosa suggests that high levels of PrpL and pyocyanin-producing isolates are more prevalent among corneal isolates and could enhance tissue damage during infection. Supporting this premise, corneal epithelial cell monolayers disrupted by high PrpL-producing strains but remained intact after treatment with P. aeruginosa mutants culture supernatants that lack or have reduced secreted PrpL.

microbiology↗

bioRxiv2024

Weakened airway epithelial junctions and enhanced neutrophil elastase release contribute to age-dependent bacteremia risk following pneumococcal pneumonia

Streptococcus pneumoniae (Sp; pneumococcus), the most common agent of community-acquired pneumonia, can spread systemically, particularly in the elderly, highlighting the need for adjunctive therapies. The airway epithelial barrier defends against bacteremia and is dependent upon apical junctional complex (AJC) proteins such as E-cadherin. After mouse lung challenge, pneumolysin (PLY), a key Sp virulence factor, stimulates epithelial secretion of an inflammatory eicosanoid, triggering the infiltration of polymorphonuclear leukocytes (PMNs) that secrete high levels of neutrophil elastase (NE), thus promoting epithelial damage and systemic infection. Here, pulmonary E-cadherin staining of intratracheally inoculated mice revealed PLY-mediated disruption of AJC independently of PMNs. Apical infection of air-liquid interface (ALI) respiratory epithelial monolayers similarly showed that PLY disrupts AJCs. This epithelial damage promoted PMN transmigration and bacterial apical-to-basolateral translocation, and pharmacologically fortifying epithelial barrier function diminished both barrier breach in vitro and bacteremia in vivo. E-cadherin staining after Sp intratracheal inoculation of >20-month-old mice, or apical infection of ALI monolayers derived from these mice, revealed an age-associated vulnerability to PLY-mediated AJC disruption, which in turn enhanced PMN migration and bacteremia. In addition, we found that PMNs from aged mice secrete increased levels of tissue-damaging NE. Simultaneous pharmacological inhibition of tissue-destructive NE and fortification of pulmonary epithelial barrier function was required to reduce the level of Sp bacteremia in aged mice to that of young mice. This work underscores the importance of fully characterizing the multifactorial sources of age-associated susceptibility in devising adjunctive therapies to mitigate invasive pneumococcal disease in the elderly.

microbiology↗

bioRxiv2024

Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins

Lyme disease is a tick-borne spirochetosis with diverse clinical manifestations. Phenotypic variation among Borrelia burgdorferi strains correlates with variable manifestations of Lyme disease in humans; this diversity is attributed in part to variation in surface-exposed lipoproteins, which are targets of the human antibody response and contribute to tissue adhesion, immune evasion, and other host interactions. Many B. burgdorferi lipoproteins are encoded as multi-copy gene families including the OspE/F- like leader peptide (Elp) protein family. To characterize Elp allelic variants, we adapted the Pseudomonas syringae ice nucleation protein (INP) system to present B. burgdorferi lipoproteins on the surface of Escherichia coli. We identified interactions with classical complement proteins and mapped binding regions in the E. coli system. We validated interactions using recombinant proteins and B. burgdorferi surface display. By assessing potential interactions with extracellular matrix components, we identified a novel interaction between Elp proteins and perlecan, a component of mammalian basement membranes, and revealed the bifunctional nature of Elps. Our findings reveal that Elps have undergone functional diversification while maintaining classical complement inhibition mediated by potent and conserved C1s binding and demonstrate that E. coli surface display offers an efficient, cost-effective, and relatively high throughput approach to characterize B. burgdorferi lipoproteins.

microbiology↗

bioRxiv2024

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr({Phi}Stx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC({Phi}Stx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC({Phi}Stx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality. Significance StatementEngineered smart microbes that secrete therapeutics are emerging as treatment modalities, particularly for gut-based diseases. With the growing threat of multidrug-resistant infection, non-antibiotic treatments are urgently needed. The gastrointestinal pathogen enterohemorrhagic E coli (EHEC) can cause the potentially lethal hemolytic uremic syndrome, a toxin-driven disease. Given concerns that antibiotics increase toxin release, treatment is largely limited to supportive care. Here, we show that pre-treatment with a commensal E. coli (HS-PROT3EcT) engineered to secrete an antibody that blocks an essential EHEC virulence factor delays the establishment of an EHEC-like infection in mice. This study strongly suggests that smart microbes that deliver payloads that block colonization factors of gut pathogens can be developed as critically needed alternatives to antibiotics for fighting bacterial infections.

microbiology↗

bioRxiv2024

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin (PLY), triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia. ImportanceStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified the eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

microbiology↗

bioRxiv2024

Streptococcus pneumoniae infection of lung epithelial cells induces internalization of surface GPI-anchored proteins through pneumolysin-mediated activation of host Rho GTPases

A return to homeostasis after infection-associated cellular injury can be accelerated by a rapid damage response. S. pneumoniae, a typically asymptomatic colonizer of the host upper respiratory tract, can cause serious and life-threatening infections when it gains access to the lungs and other organs. The cholesterol binding S. pneumoniae pore-forming toxin, pneumolysin (PLY), is central to the induction of host cell damage. Here, we first found that mouse lung infection by S. pneumoniae diminished pulmonary expression of CD73, a glycosylphosphatidylinositol anchored protein (GPI-AP) that modulates inflammation. Infection of the human pulmonary epithelial cell line H292 resulted in a PLY-dependent reduction of not only cell surface CD73, but also the population of surface expressed GPI-APs. The decrease in cell surface GPI-APs was rapid, required pore-forming activity, and could be recapitulated by purified PLY and other cholesterol binding cytolysins. In response to PLY-mediated insult, GPI-APs were not released from the surface of epithelial cells in extracellular vesicles but rather internalized by a mechanism dependent on the Rho GTPases RhoA and Cdc42. Internalization of GPI-APs was associated with lower levels of PLY-induced apoptosis and membrane permeabilization. These findings suggest that internalization of GPI-APs from epithelial cell membranes may constitute a rapid innate repair response to cell damage induced by PLY and other pore forming toxins that could help bacteria evade host defenses as many GPI-APs have roles in immunity. Author summaryStreptococcus pneumoniae causes serious infections that can result in mortality. The pore- forming toxin, pneumolysin (PLY) produced by these bacteria is important for their ability to cause disease. Understanding how the host responds to damage by this toxin can result in better treatment against infection. In this study, we found that PLY-mediated injury results in decreased expression of glycosylphosphatidylinositol anchored proteins (GPI-AP) from the cell surface by internalization. GPI-AP co-localize in cholesterol-rich areas of the membrane where PLY inserts to form pores and cells with decreased surface GPI-APs were associated with less of PLY-induced cell death and membrane permeabilization. These results suggest that GPI-AP are internalized as part of repair mechanisms activated in response to infection-induced cell injury. As many GPI-APs have important roles in the immune response, their removal from the cell may inadvertently help the bacteria establish better infection.

microbiology↗

bioRxiv2023

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and PLY-independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

microbiology↗

bioRxiv2023

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto (Bbss) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of [~]800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

microbiology↗

bioRxiv2022

Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family - termed ElpB and ElpQ - that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using surface plasmon resonance, Alpha bead-based technology, and C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks C4 cleavage by competing with C1s/C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp/C1s binding, we performed site-directed mutagenesis which showed that ElpB- and ElpQ-binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

biochemistry↗

bioRxiv2021

Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

Spirochetal pathogens such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria such as Proteobacteria and Firmicutes, very few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least eighty surface-exposed lipoproteins throughout its tick vector-vertebrate host lifecycle (57). Several B. burgdorferi lipoproteins have been shown to serve diverse roles, such as cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-mediated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ErpB and ErpQ. Using biochemical, microbiological, and biophysical approaches, we demonstrated that ErpB and ErpQ inhibit the activated forms of the C1 proteases, C1r and C1s, and represent a new mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing by allosteric regulation. In addition to identifying a novel mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host-pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent. Significance StatementSpirochetal pathogens encode an abundance of lipoproteins that can provide a critical interface with the host environment. For example, Borrelia burgdorferi, the model species for spirochetal biology, must survive an enzootic life cycle defined by fluctuations between vector (tick) and vertebrate host. While B. burgdorferi expresses over eighty surface lipoproteins-- many of which likely contribute to host survival--the B. burgdorferi lipoproteome is poorly characterized. Here, we generated a platform to rapidly identify targets of B. burgdorferi surface lipoproteins and identified two orthologs that allosterically inhibit complement C1 subcomponents, conferring resistance to classical complement killing. This work expands our understanding of complement evasion mechanisms and points towards a discovery approach for identifying host-pathogen interactions that are central to spirochete pathogenesis.

microbiology↗

bioRxiv2021

Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic Escherichia.coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eucaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics. Author SummaryEnteric pathogens are the causative agents of diarrheal disease - a leading cause of infant morbidity and mortality worldwide. While treatment and prevention options such as drugs or vaccines exist for some pathogens, their efficacy and availability are often limited. New therapeutic strategies are therefore needed, especially inexpensive agents in low-income countries where enteric disease burdens are highest. One promising avenue for novel treatments uses VHHs - highly stable, well-expressed, antibody domains derived from camelid species such as llamas and alpacas. The small size, high stability and simple structure of these antibody fragments enables their streamlined production by bacteria such as E. coli, potentially reducing cost and improving scalability. In this work, we describe the development of VHHs targeting multiple virulence factor proteins of pathogenic E. coli and other leading causes of diarrheal disease. These VHHs provide new tools for the research community and may serve as promising components of agents that prevent or treat pathogen infections. Towards that goal, we engineered a novel system in which the probiotic, mucus-establishing bacterial strain E. coli Nissle 1917 (EcN) is used to express and display VHHs at high density on its surface. By demonstrating the ability of these engineered EcN to bind to pathogens, we provide a first step toward using such probiotics as a cheap, simple, and effective treatment for enteric pathogen infections.

microbiology↗

bioRxiv2020

A Murine Model for the Transition of Streptococcus pneumoniae from Asymptomatic Colonizer to Systemic Pathogen

Streptococcus pneumoniae (pneumococcus) resides asymptomatically in the nasopharynx but can progress from benign colonizer to lethal pulmonary or systemic pathogen. Both viral infection and aging are risk factors for serious pneumococcal infections. Previous work established a murine model that featured the movement of pneumococcus from the nasopharynx to the lung upon nasopharyngeal inoculation with influenza A virus (IAV) but did not fully recapitulate the severe disease associated with human co-infection. We built upon this model by first establishing pneumococcal nasopharyngeal colonization, then inoculating both the nasopharynx and lungs with IAV. In young (2 months) mice, co-infection triggered bacterial dispersal from the nasopharynx into the lungs, pulmonary inflammation, disease and mortality in a fraction of mice. In old mice (20-22 months), co-infection resulted in earlier and more severe disease. Aging was not associated with greater bacterial burdens but rather with more rapid pulmonary inflammation and damage. Both aging and IAV infection led to inefficient bacterial killing by neutrophils ex vivo. Conversely, aging and pneumococcal colonization also blunted IFN- production and increased pulmonary IAV burden. Thus, in this multistep model, IAV promotes pneumococcal pathogenicity by modifying bacterial behavior in the nasopharynx, diminishing neutrophil function, and enhancing bacterial growth in the lung, while pneumococci increase IAV burden likely by compromising a key antiviral response. Thus, this model provides a means to elucidate factors, such as age and co-infection, that promote the evolution of S. pneumoniae from asymptomatic colonizer to invasive pathogen, as well as to investigate consequences of this transition on antiviral defense.

microbiology↗

bioRxiv2019

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

PMNs are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the anti-microbial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its anti-microbial activity by inhibiting IL-10 production. PMNs from wild type mice did not increase IL-10 production in response to S. pneumoniae, however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of wild type PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake and intracellular killing or production of anti-microbial Neutrophil Elastase and Myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal ROS production by PMNs. ROS contributed to PMN anti-microbial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN anti-microbial phenotype during S. pneumoniae infection.

immunology↗

bioRxiv2019

A1 Adenosine Receptor Signaling Reduces Streptococcus pneumoniae Adherence to Pulmonary Epithelial Cells by Targeting Expression of Platelet-Activating Factor Receptor

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host-pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet-activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community-acquired pneumonia in the elderly, we explored the role of A1 in the age-driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial-host interactions.

immunology↗

bioRxiv2018

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice.\n\nSome commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacterrodentium lysogenized with the Stx2-encoding phage {Phi}stx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that {Phi}stx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium ({Phi}stx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium ({Phi}stx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.\n\nAuthor summaryEnterohemorrhagic Escherichia coli (EHEC), a food-borne pathogen that produces Shiga toxin, is associated with serious disease outbreaks worldwide, including over 390 food poisoning outbreaks in the U.S. in the last two decades. Humans acquire EHEC by ingesting contaminated food or water, or through contact with animals or their environment. Infection and toxin production may result in localized hemorrhagic colitis, but may progress to life-threatening systemic hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children. Treatment for EHEC or HUS remains elusive, as antibiotics have been shown to exacerbate disease.\n\nShiga toxin genes reside on a dormant bacterial virus present in the EHEC genome, but are expressed when the virus is induced to leave its dormant state and begin to replicate. Extensive virus replication has been thought necessary to produce sufficient toxin to cause disease.\n\nUsing viral and bacterial mutants in our EHEC disease mouse model, we showed that whereas an inducing signal needed to begin viral replication was essential for lethal disease, virus production was not: sufficient Shiga toxin was produced to cause lethal mouse disease, even without viral replication. Future analyses of EHEC-infected human samples will determine whether this same phenomenon applies, potentially directing intervention strategies.

microbiology↗