Biology subjects

Castellino, F. J.

Publications and source records attributed to Castellino, F. J..

8 recordsLinked to original sources

bioRxiv2024

Coagulation factor XII contributes to renin activation, heart failure progression, and mortality

Symptomatic heart failure (sHF) with cardiac dysfunction, edema, and mortality are driven by overactivation of the renin-angiotensin-aldosterone system (RAAS). Renin is widely recognized as a key initiator of RAAS function, yet the mechanisms that activate renin remain a mystery. We discovered that activated coagulation factor XII generates active renin in the circulation and is directly linked to pathological activation of the systemic RAAS, development of sHF, and increased mortality. These findings suggest a new paradigm for therapeutically modulating the RAAS in sHF and other pathological conditions.

pathology↗

bioRxiv2024

The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI

Urinary catheterization causes bladder damage, predisposing hosts to catheter-associated urinary tract infections (CAUTIs). CAUTI pathogenesis is mediated by bladder damage-induced inflammation, resulting in accumulation and deposition of the blood-clotting protein fibrinogen (Fg) and its matrix form fibrin, which are exploited by uropathogens as biofilm platforms to establish infection. Catheter-induced inflammation also results in robust immune cell recruitment, including macrophages (M{phi}s). A fundamental knowledge gap is understanding the mechanisms by which the catheterized-bladder environment suppresses the M{phi} antimicrobial response, allowing uropathogen persistence. Here, we found that Fg and fibrin differentially modulate M1 and M2 M{phi} polarization, respectively. We unveiled that fibrin accumulation in catheterized mice induced an anti-inflammatory M2-like M{phi} phenotype, correlating with pathogen persistence. Even GM-CSF treatment of wildtype mice to promote M1 polarization was not sufficient to reduce bacterial burden and dissemination, indicating that the catheterized-bladder environment provides mixed signals, dysregulating M{phi} polarization, hindering its antimicrobial response against uropathogens.

immunology↗

bioRxiv2023

Group A Streptococcus remains viable inside fibrin clots and gains access to human plasminogen for subsequent fibrinolysis and dissemination

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a wide spectrum of illnesses ranging from pharyngitis and rheumatic fever to more invasive and severe diseases such as necrotizing fasciitis and toxic shock syndrome. Invasive outcomes of GAS infections often result from entry of the bacteria via an open wound into tissue and blood systems. The coagulation cascade serves as an innate defense mechanism that initiates fibrin clots to sequester bacteria and restrict its growth and prevent dissemination into deeper tissues. GAS, especially skin-tropic bacterial strains, utilize the specific virulence factors Plasminogen binding M-protein (PAM) and streptokinase (SK) to manipulate hemostasis and ultimately activate human plasminogen to cause fibrinolysis and escape from the fibrin clot. A major unresolved question regarding this process is to understand the temporal dynamics of how GAS that is enmeshed in a fibrin clot accesses host plasminogen for dissolution of the clot and eventual dissemination. Using fluorescently labeled plasminogen and fibrinogen, we established conditions to observe the process of fibrin clot dissolution by GAS (an AP53 CovR+S-strain) that is sequestered in a fibrin clot using real-time imaging microscopy. We hypothesized that initiation of fibrinolysis by GAS inside a fibrin clot would be determined by the rate of hPg access into the fibrin clot where bacteria are trapped. Our live imaging studies show that GAS trapped inside a fibrin clot, has limited access to hPg; however, at 4.25 h post incubation, when sufficient hPg is accessible to the bacterium, fibrinolysis quickly occurs. If hPg is bound to the bacterial surface prior to being trapped inside a clot, dissolution and bacterial dissemination occurs at a much faster rate of 2.5 h post incubation. During the time which bacteria are trapped in the clot without access to hPg, we did not observe any growth of GAS; however, we demonstrate that the bacteria continue to remain viable inside the fibrin clot. We performed RNA-seq analysis of GAS and the isogenic GAS SK-deficient mutant to understand SK-dependent transcriptional changes during the lag-phase of the GAS bacteria inside the fibrin clot. We observed a dramatic change in the transcription profile of wt GAS inside the fibrin clot over time prior to escape from the fibrin clot (22 gene expression changes at 4h, to 802 gene expression changes at 8h). Furthermore, we also identified gene expression changes that were distinct between wt GAS and the GAS SK-deficient mutant. Our findings reveal for the first time that GAS can engage a latent, growth suspended phase whereby physical structures such as fibrin clots and Neutrophil extracellular traps that immobilize an invading pathogen allow bacteria to remain viable and transcriptionally active for an extended time during host infection. GAS that is trapped in a fibrin clot will therefore enter a state in which the bacteria suspend growth, but remain viable, until sufficient access to hPg allow it to initiate fibrinolysis and escape into surrounding tissues. The viability of GAS while trapped and its readiness to avoid immune defenses allow GAS to act quickly to disseminate when host conditions are more favorable for the bacteria.

microbiology↗

bioRxiv2023

A high-resolution cryo-EM structure of a bacterial M-protein reveals a compact structure that diverges from related M-proteins

The surface of Streptococcus pyogenes (GAS) is studded with virulence determinants, with the most abundant being the characteristic M-protein used to serotype various strains of the bacterium. There are >250 strains of GAS serotypically distinguished by their M-proteins. Major pathogenic mechanisms of GAS require that this microorganism hijacks host components for survival, many of which are involved in hemostasis. One of these processes involves the binding of human host plasminogen (hPg) to an abundant GAS M-protein receptor (PAM). When bound to PAM, hPg is readily activated to the serine protease plasmin (hPm) by bacterial and host hPg activators, and cell-bound hPm is protected from inactivation by its natural inhibitors. This stabilizes a potent protease on GAS cells which aids in their survival and dissemination. Highly evolutionary domain-related M-proteins are assumed to form long alpha-helical projections, without tertiary structure, although no M-protein complete structure has been determined. Here, we employed cryogenic electron microscopy to solve such a structure anchored to a lentivirus particle membrane. Contrary to the belief in this field that M-proteins are extended long tropomyosin-like coils, we show that PAM folds through intra- and inter-domain interactions to a much more globular form on the cell surface. The nature of the folding and the many interactions involved in forming the PAM tertiary structure are summarized herein. SignificanceWe provide a unique approach to solve high-resolution structures of Streptococcus pyogenes (GAS) M-proteins, abundant virulence determinants on the GAS surface. Because of their unusual nature, no full high-resolution structure of any M-protein has been determined, especially when membrane-bound. Herein, we provide a unique general methodology for solving these structures by engineering a M-protein to be anchored to a lentivirus particle membrane for effective use in cryo-EM. Using this approach, we provide the first structure of a complete bacterial M-protein and show, that this M-protein is a monomeric globular structure on the cell surface, and not a dimeric coiled-coil, as generally believed. Thus, individual M-proteins may adopt structures that have evolved to accommodate their major host binding partner.

biochemistry↗

bioRxiv2022

A high-resolution single-particle cryo-EM hydrated structure of Streptococcus pyogenes enolase offers insights into its function as a plasminogen receptor

Cellular plasminogen (Pg) receptors (PgR) are utilized to recruit Pg, stimulate its activation to the serine protease, plasmin (Pm), and sterically protect the generated Pm from inactivation by natural host inhibitors. The net result is that cells contain a stable proteolytic surface used for biological mechanisms involved in cell migration. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this regard. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 [A] as a stable symmetrical homooctamer displaying point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ~49 kDa. Subunit-subunit interactions showed four major and four minor interfaces in the octamer. Binding sites for hPg were previously proposed to include the COOH-terminal K434,435 residues of Sen, but in native Sen these residues are buried within the minor interfaces of the octamer and do not function as a Pg binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K434,434. We propose that the octameric structure of Sen is important to its ability to interact with hPg, but disruption of its overall octameric conformation without dissociation of the octamer exposes neoepitopes for hPg binding.

biochemistry↗

bioRxiv2021

Evolution of Gram+ Streptococcus pyogenes has maximized efficiency of the Sortase A cleavage site

Human plasminogen (hPg)-binding M-protein (PAM), a major virulence factor of Pattern D Streptococcus pyogenes (GAS), is the primary receptor responsible for binding and activating hPg. PAM is covalently bound to the cell wall (CW) through cell membrane (CM)-resident sortase A (SrtA)-catalyzed cleavage of the PAM-proximal C-terminal LPST{downarrow}-GEAA motif present immediately upstream of its transmembrane domain (TMD), and subsequent transpeptidation to the CW. These steps expose the N-terminus of PAM to the extracellular milieu (EM) to interact with PAM ligands, e.g., hPg. Previously, we found that inactivation of SrtA showed little reduction in functional binding of PAM to hPg, indicating that PAM retained in the cell membrane (CM) by the TMD nonetheless exposed its N-terminus to the EM. In the current study, we assessed the effects of mutating the Thr4 (P1) residue of the SrtA-cleavage site in PAM (Thr355 in PAM) to delay PAM in the CM in the presence of SrtA. Using rSrtA in vitro, LPSYGEAA and LPSWGEAA peptides were shown to have low activities, while LPSTGEAA had the highest activity. Isolated CM fractions of AP53/{Delta}SrtA cells showed that LPSYGEAA and LPSWGEAA peptides were cleaved at substantially faster rates than LPSTGEAA, even in CMs with an AP53/{Delta}SrtA/PAM[T355Y] double mutation, but the transpeptidation step did not occur. These results implicate another CM-resident enzyme that cleaves LPSYGEAA and LPSWGEAA motifs, most likely LPXTGase, but cannot catalyze the transpeptidation step. We conclude that the natural P1 (Thr) of the SrtA cleavage site has evolved to dampen PAM from nonfunctional cleavage by LPXTGase. IMPORTANCEWe show in this study that functional cleavage of the sortase A (SrtA) cleavage signal for M-protein, LPST*GEAA, in the Gram+ cell membrane, which allows transpeptidation of M-protein to the cell wall, as opposed to non-functional cleavage by the highly active cell membrane nonribosomal enzyme, LPXTGase, at the downstream G-residue, is highly dependent on the presence of T at position 4. From our studies, we conclude that Streptococcus pyogenes has evolved in a manner that maximized T at this position so that SrtA preferentially cleaved the sorting signal in order that the virulence factor, M-protein, was stabilized on the cell surface through covalent attachment to the cell wall.

microbiology↗

bioRxiv2021

Fibrin is a critical regulator of neutrophil effector function at mucosal barrier sites

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we document that the clotting factor fibrin is a critical regulator of neutrophil function at mucosal barriers. We demonstrate that fibrin engages neutrophils through the M{beta}2 integrin receptor and activates effector functions, including the production of reactive oxygen species and NET formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis. Indeed, the accumulation of fibrin due to Mendelian genetic defects in plasmin leads to severe oral mucosal immunopathology in mice and humans. Concordantly, genetic polymorphisms in the human PLG gene, encoding plasminogen, are associated with common forms of the oral mucosal disease periodontitis. Our work uncovers fibrin as a critical regulator of neutrophil effector function within the mucosal tissue microenvironment and suggests fibrin-neutrophil engagement as a pathogenic instigator and therapeutic target in common mucosal disease.

immunology↗

bioRxiv2019

Discovery of genes encoding a Streptolysin S-like toxin biosynthetic cluster in a select highly pathogenic methicillin resistant Staphylococcus aureus JKD6159 strain

BackgroundStaphylococcus aureus (S. aureus) is a major human pathogen owing to its arsenal of virulence factors, as well as its acquisition of multi-antibiotic resistance. Here we report the identification of a Streptolysin S (SLS) like biosynthetic gene cluster in a highly virulent community-acquired methicillin resistant S. aureus (MRSA) isolate, JKD6159. Examination of the SLS-like gene cluster in JKD6159 shows significant homology and gene organization to the SLS-associated biosynthetic gene (sag) cluster responsible for the production of the major hemolysin SLS in Group A Streptococcus.\n\nResultsWe took a comprehensive approach to elucidating the putative role of the sag gene cluster in JKD6159 by constructing a mutant in which one of the biosynthesis genes (sagB homologue) was deleted in the parent JKD6159 strain. Assays to evaluate bacterial gene regulation, biofilm formation, antimicrobial activity, as well as complete host cell response profile and comparative in vivo infections in Balb/Cj mice were conducted.\n\nConclusionsAlthough no significant phenotypic changes were observed in our assays, we postulate that the SLS-like toxin produced by this strain of S. aureus may be a highly specialized virulence factor utilized in specific environments for selective advantage; studies to better understand the role of this newly discovered virulence factor in S. aureus warrant further investigation.

microbiology↗