Zhe Ma

Advances in microscopy have enabled the widespread application of in vivo pathogen tracking approaches to bacterial pathogenesis. Pasteurella multocida ( P. multocida ) is a major zoonotic pathogen causing severe respiratory diseases in livestock. However, genetic manipulation of clinical swine-derived isolates remains hindered by the lack of stable genetic tools. As a result, no genetically engineered in vivo tracking strain limits research in P. multocida infection dynamics. In this study, we evaluated diverse replication mechanisms and identified that rolling circle replication (RCR)-type replicons exhibit superior transformation robustness in swine isolates. Leveraging the temperature-sensitive pSET4s backbone and an optimized mpheS counter-selection marker, we developed a high-efficiency, markerless genetic manipulation system. The system exhibited high copy numbers and significantly enhanced transformation yields across diverse clinical swine isolates. To validate gene knockout efficiency, we performed markerless deletions of the capsule biosynthesis genes hyaD and hyaE . Phenotypic characterization revealed that hyaluronic acid (HA) deficiency led to complete attenuation of virulence in a murine model. Furthermore, we engineered a 15-Pm::GFP reporter strain via site-specific gene knock-in without detectable effects on bacterial growth or virulence. This strain enables direct visualization of bacterial colonization in the lung and liver by fluorescence imaging and allows real-time, in situ monitoring of pathogen and Kupffer cell interactions. Collectively, our results established an effective genetic toolkit for swine-derived P. multocida and developed fluorescently labeled strains for in vivo tracking. This plasmid provides a useful tool for investigating pathogenic mechanisms and in vivo infection dynamics of P. multocida . IMPORTANCE: P. multocida is a critical threat to global animal health, while the difficulty of genetic modification in swine isolates has interrupted the link of the genomic and pathogenic phenotypes. This study provides a highly efficient, RCR-based markerless manipulation system optimized with cross-serogroup applicability for swine isolates. Validated across 15 clinical swine isolates, this plasmid offers a robust tool for genetic analysis and the rational design of next-generation vaccines. By defining the essential roles of hyaD and hyaE , we demonstrate that the hyaluronic acid capsule is required for virulence and pulmonary colonization. Moreover, the development of a biologically fluorescent reporter strain enables high-resolution, in situ tracking of infection dynamics, providing a powerful tool for real-time visualization during P. multocida infection.

Streptococcal peritonitis is associated with a high mortality risk and sometimes accompanied by streptococcal toxic shock-like syndrome (STSLS). Here, we used a murine intraperitoneal model of Streptococcus suis serotype 2 (SS2) infection and applied bacterial lineage tracing to investigate the pathogen’s population dynamics during streptococcal intraperitoneal infection. We found that the cholesterol-dependent cytolysin suilysin (SLY) was essential for acute lethality and facilitated bacterial systemic spread by mitigating infection bottlenecks and enabling vascular leakage in the peritoneal cavity. SLY induced marked accumulation of arachidonic acid (AA) in the peritoneal cavity. Although inhibition of AA synthesis alone did not prevent mortality, it widened the therapeutic window of antibiotic treatment, enhancing their efficacy and capacity to reduce lethality. These findings identify SLY as a critical virulence determinant linking inflammation-associated metabolic alterations, disruption of vascular integrity, and mortality and highlight AA metabolism as a promising adjunctive therapeutic target for acute SS2 intraperitoneal infection.

As a zoonotic opportunistic pathogen, adhesion to the porcine upper respiratory tract epithelium is a prerequisite for Streptococcus suis serotype 2 (SS2) to breach epithelial barriers and cause systemic infections under certain conditions. In this study, we first demonstrated the adhesive function of the autolysin Atl of SS2 to swine tracheal epithelial cells. We found that Atl functions as a molecular bridge for epithelial adhesion. The Bsp-like domain (Atl ^Bsp ) specifically binds bacterial lipoteichoic acid, anchoring to the bacterial surface, while the C-terminal hydrolase domain (Atl ^COOH ) directly interacts with host epithelial receptor Fibronectin (Fn). With the purified recombinant Atl (rAtl), we demonstrated that both membrane-bound and secreted forms of Atl could mediate adhesion to the epithelial cells. Moreover, the rAtl was able to restore the adhesive capacity of the Atl-deficient SS2 mutant (Δ atl ) to both porcine and murine upper respiratory tract epithelium. This study reveals a novel function of the autolysin Atl in promoting SS2 adhesion to the upper respiratory tract epithelium, which elucidates the underlying molecular mechanism and identifies the key functional domains involved. These findings offer new insights into autolysin biology and provide a theoretical basis for developing anti-adhesion strategies against streptococcal infections in pigs.

Proliferation of the emerging zoonotic pathogen Streptococcus equi subsp. zooepidemicus in the meninges is linked to mortality in pigs and morbidity in humans. The mechanisms underlying the remarkable capacity of hypervirulent S. zooepidemicus to proliferate in cerebrospinal fluid (CSF) are largely undefined. Here, using genetically barcoded S. zooepidemicus , we found that following systemic infection of mice, only ~1–10 S. zooepidemicus clones invade the meninges where they subsequently replicate ~10 ⁷ -fold. Subsequent transposon insertion sequencing experiments, plus validation work with bacterial mannose phosphotransferase system (PTS _man )-defective strains, identified the PTS _man , which imports glucose, as essential for S. zooepidemicus proliferation in CSF. The S. zooepidemicus PTS _man promoter confers species-specific constitutive transcription of PTS _man , enabling glucose acquisition at low glucose concentrations and limiting activation of the stringent response, leading to pathogen replication in CSF. Our findings reveal how the rewiring of PTS _man in the control of S. zooepidemicus metabolism enables this pathogen to adapt to and replicate in CSF during meningitis.

Streptococcus suis serotype 2 (SS2) infection causes streptococcal toxic shock-like syndrome (STSLS), which can be lethal in both pigs and humans. In mouse models, relatively subtle differences in the inoculum size result in drastically different infection outcomes, from clearance to acute death. Here, we leveraged barcoded SS2 in a murine model to define the bacterial population dynamics associated with death. Barcode sequencing analysis demonstrated that a 10-fold increase in inoculum size led to a 100-1,000-fold increase in the number of bacteria that initiated infection in certain lymph nodes, ultimately driving lethality. This lethal dose threshold was in part driven by suilysin (SLY), the major SS2 virulence factor. At higher doses, the clearance and replication kinetics of bacteria lacking SLY mimicked WT SS2 at sub-lethal doses. Our findings suggest that acute death in SS2 infection is caused by a wholesale failure of the immune system, enabling a large number of bacteria to initiate infection. These results demonstrate how subtle changes in inoculum size can have dramatic consequences for infection outcome and how lethal infection is driven by widened infection bottlenecks. Author Summary: Lethal infections are often associated with a specific dose threshold, where increasing inoculum size is associated with an increased likelihood of death. However, a quantitative explanation how this numeric threshold is established is lacking. For example, lethal dose thresholds may reflect a dose where at least one microbe can pass through host-imposed barriers to infection. Alternatively, lethal dose thresholds may reflect a failure of the host immune system, leading to numerous microbes initiating infection. Here, using barcoded Streptococcus suis serotype 2 (SS2), we compare the within-host population dynamics associated between lethal and non-lethal infections. We find that lethal doses are associated with dramatic widening of infection bottlenecks, leading to a large number of bacteria that initiate infection. Lethal infection is also driven by suilysin, the major SS2 virulence factor. These observations demonstrate how lethal doses are quantitatively established by the relationship between host bottlenecks and bacterial virulence factors.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.