Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

Detection of Porcine Circovirus Type 3 in Serum, Semen, Oral Fluid, and Preputial Fluid Samples of Boars.

Porcine circovirus type 3 (PCV3) is commonly associated with clinical symptoms such as porcine dermatitis and nephropathy syndrome (PDNS)-like lesions, respiratory signs, and reproductive disorders. This study aimed to investigate the epidemiology of PCV3 in a boar stud. The objectives were to detect PCV3 in semen, as well as matched serum, oral fluid, and preputial fluid samples from adult boars using quantitative polymerase chain reaction (qPCR), analyze PCV3-IgG antibody data, and genetically characterize a positive sample. A total of 112 samples from 28 boars were collected from a large-scale pig farm in Guangxi, China. The qPCR results showed that the PCV3 DNA was not detected in semen, with a positive rate of 0% (0/28), while it was detected in serum (3.57%-1/28), oral fluid (64.28%-18/28), and preputial fluid (46.4%-13/28). The seropositivity rate of PCV3-IgG in serum was 82.14% (23/28) according to the indirect enzyme-linked immunosorbent serologic assay (ELISA) results. Phylogenetic analysis revealed that one of the PCV3 isolates belonged to the PCV3c clades. This is the first report of PCV3 detection in preputial fluid from boars. The results suggest that PCV3 is transmitted among boars on pig farms and exhibits epidemic characteristics.

Effects of combined application of benzoic acid and 1-monolaurin on growth performance, nutrient digestibility, gut microbiome and inflammatory factor levels in weaned piglets.

BACKGROUND: Our previous study observed that benzoic acid and 1-monolaurin have a synergistic bactericidal effect. Moreover, their improvement effect of benzoic acid and 1-monolaurin on the growth performance and diarrhea of weaned piglets was better than the two feedings alone. However, it is not clear how the combination of benzoic acid and 1-monolaurin affects the growth performance of weaned piglets. Therefore, 100 weaned piglets (mean weight 7.03 ± 1.04 kg, mean weaning age 26 d) were randomly divided into two groups: (1) basal diet control (CON); (2) basal diet supplemented with 0.6% benzoic acid and 0.1% 1-monolaurin (CA). The experiment lasted 28 days after weaning. The effects of benzoic acid and 1-monolaurin supplementation on growth performance, apparent nutrient digestibility, intestinal flora composition and function, and inflammatory factor levels of weaned piglets were investigated. RESULTS: The feed conversion efficiency of piglets in the CA group between 15 and 28 d and 1 and 28 d after weaning was significantly higher than that in the CON group (P < 0.05). Additionally, the diarrhea proportion and frequency of piglets in the CA group 1-14 days post-weaning were significantly decreased (P < 0.05). The apparent digestibility of dry matter, organic matter and crude protein of piglets in the CA group was significantly higher than the CON group on days 14 and 28 (P < 0.05). The microbial composition in the cecal digesta of piglets was detected. The results indicated that the CA group piglets were significantly supplemented with g_YRC22 at day 14 and g_Treponema, g_Pseudomonas, and g_Lachnobacterium at day 28 (P < 0.05; log LDA > 2). No significant difference was observed between the CON and CA groups in the content of short-chain fatty acids. In addition, serum IL-1ß level significantly decreased at day 28 in the CA group compared with the CON group, while serum endotoxin content was significantly reduced at day 14. CONCLUSION: Therefore, dietary supplementation of 0.6% benzoic acid and 0.1% 1-monolaurin enhanced growth performance and nutrient digestibility, affected gut microflora composition, and decreased systemic inflammatory response and intestinal permeability of weaned piglets. These outcomes provide a theoretical basis for applying of benzoic acid and 1-monolaurin over weaned piglets.

The two-component system CpxAR is required for the high potassium stress survival of Actinobacillus pleuropneumoniae.

Introduction: Actinobacillus pleuropneumoniae is an important respiratory pathogen, which can cause porcine contagious pleuropneumonia and lead to great economic losses to worldwide swine industry. High potassium is an adverse environment for bacteria, which is not conducive to providing turgor pressure for cell growth and division. Two-component system CpxAR is an important regulatory system of bacteria in response to environmental changes, which is involved in a variety of biological activities, such as antibiotic resistance, periplasmic protein folding, peptidoglycan metabolism and so on. Methods: However, little is known about the role of CpxAR in high potassium stress in A. pleuropneumoniae. Here, we showed that CpxAR is critical for cell division of A. pleuropneumoniae under high potassium (K+) stress. Results: qRT-PCR analysis found that CpxAR positively regulated the cell division genes ftsEX. In addition, we also demonstrated that CpxR-P could directly bind the promoter region of the cell division gene ftsE by EMSA. Discussion: In conclusion, our results described a mechanism where CpxAR adjusts A. pleuropneumoniae survival under high-K+ stress by upregulating the expression of the cell division proteins FtsE and FtsX. These findings are the first to directly demonstrate CpxAR-mediated high-K+ tolerance, and to investigate the detailed molecular mechanism.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

The adcA and lmb Genes Play an Important Role in Drug Resistance and Full Virulence of Streptococcus suis.

Streptococcus suis is an recognized zoonotic pathogen of swine and severely threatens human health. Zinc is the second most abundant transition metal in biological systems. Here, we investigated the contribution of zinc to the drug resistance and pathogenesis of S. suis. We knocked out the genes of AdcACB and Lmb, two Zn-binding lipoproteins. Compared to the wild-type strain, we found that the survival rate of this double-mutant strain (ΔadcAΔlmb) was reduced in Zinc-limited medium, but not in Zinc-supplemented medium. Additionally, phenotypic experiments showed that the ΔadcAΔlmb strain displayed impaired adhesion to and invasion of cells, biofilm formation, and tolerance of cell envelope-targeting antibiotics. In a murine infection model, deletion of the adcA and lmb genes in S. suis resulted in a significant decrease in strain virulence, including survival rate, tissue bacterial load, inflammatory cytokine levels, and histopathological damage. These findings show that AdcA and Lmb are important for biofilm formation, drug resistance, and virulence in S. suis. IMPORTANCE Transition metals are important micronutrients for bacterial growth. Zn is necessary for the catalytic activity and structural integrity of various metalloproteins involved in bacterial pathogenic processes. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. Thus, pathogenic bacteria must acquire Zn during infection in order to successfully survive and multiply. The host uses nutritional immunity to limit the uptake of Zn by the invading bacteria. The bacterium uses a set of high-affinity Zn uptake systems to overcome this host metal restriction. Here, we identified two Zn uptake transporters in S. suis, AdcA and Lmb, by bioinformatics analysis and found that an adcA and lmb double-mutant strain could not grow in Zn-deficient medium and was more sensitive to cell envelope-targeting antibiotics. It is worth noting that the Zn uptake system is essential for biofilm formation, drug resistance, and virulence in S. suis. The Zn uptake system is expected to be a target for the development of novel antimicrobial therapies.

Novel Multiplex PCR Assay and Its Application in Detecting Prevalence and Antibiotic Susceptibility of Porcine Respiratory Bacterial Pathogens in Guangxi, China.

Porcine respiratory disease complex (PRDC) is a serious disease caused by multiple pathogens which inflicts huge economic losses on the pig industry. Investigating the epidemiology of porcine respiratory bacterial pathogens (PRBPs) in specific geographic areas and exploring the antibiotic susceptibility of local strains will contribute to the prevention and control of PRDC. However, the epidemiology of PRBPs in Guangxi Province remains unclear, and existing diagnostic methods have multiple limitations, such as high costs and the detection of only a single pathogen at a time. In this study, we developed a multiplex PCR assay for Streptococcus suis, Glaesserella parasuis, Actinobacillus pleuropneumoniae, Pasteurella multocida, and Mycoplasma hyopneumoniae, and investigated the prevalence of PRBPs in pigs with respiratory symptoms in Guangxi Province. The isolates from positive samples were subjected to susceptibility tests to 16 antibiotics. Our results indicated that of the 664 samples from pigs with respiratory symptoms, 433 (65.21%), 320 (48.19%), 282 (42.47%), 23 (3.46%), and 9 (1.36%), respectively, carried each of these 5 pathogens; 533 samples were positive; and 377 (56.78%) carried multiple pathogens simultaneously. The dominant PRBPs in pigs with respiratory symptoms in Guangxi province were S. suis, G. parasuis, and A. pleuropneumoniae, which frequently co-infected swine herds. Most of the isolates (A. pleuropneumoniae, G. parasuis, S. suis, and P. multocida) were sensitive to cefquinome, ceftiofur, trimethoprim-sulfamethoxazole (TMP-SMX), and tiamulin antibiotics. We developed a rapid specific multiplex PCR assay for PRBPs. Our findings provide new information on the epidemiology of PRBPs in Guangxi Province and offer a reference for developing drug targets against PRDC. IMPORTANCE Pigs are closely associated with humans as the most common food animals and the vectors of numerous pathogens. PRDC, caused by multiple pathogens, is a serious disease that can cause growth retardation in swine and even sudden death. Due to the droplet transmission of PRBP and the similar clinical signs of different pathogen infections, most pig farms struggle to identify and control PRBPs, leading to the abuse of antibiotics. In addition, some PRBPs have the potential to infect humans and threaten human health. Therefore, this study developed a multiplex PCR method targeting PRBPs, investigated the prevalence of these pathogens, and tested their antibiotic susceptibility. Our studies have important implications for public health safety and the development of the pig industry.

Mutations in troABCD against Copper Overload in a copA Mutant of Streptococcus suis.

Streptococcus suis is a major swine pathogen that is increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role during the process of bacterial infection. In this study, RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular copper homeostasis. CopA was identified as the primary copper exporter in S. suis. The copA deletion mutant strain was found to be more sensitive to copper and accumulated more intracellular copper than the wild-type (WT) parent strain. In addition, adding manganese increased the ability of S. suis to resist copper, and the manganese transporter, TroABCD, was involved in tolerance to copper. The copA deletion mutant strain accumulated less copper when supplemented with manganese. Furthermore, when cultured with copper, the double deletion mutant (ΔcopAΔtroA) exhibited improved growth compared to the copA deletion mutant strain. In addition, the double deletion mutant (ΔcopAΔtroA) accumulated less copper than the copA deletion mutant strain. These data were consistent with a model wherein defective TroABCD resulted in decreased cellular copper accumulation and protected the strain against copper poisoning. IMPORTANCE Metal homeostasis plays a critical role during the process of bacterial infection. We identified three important potential candidate genes involved in maintenance of intracellular copper homeostasis. CopA was demonstrated to be the main copper exporter in Streptococcus suis, and manganese increased the tolerance of S. suis to copper. The double deletion mutant (ΔcopAΔtroA) improved growth ability over the copA deletion mutant strain in the presence of high concentrations of copper and accumulated less copper. These findings are consistent with a model wherein defective TroABCD resulted in decreased cellular accumulation of copper and protected the strain against copper poisoning.

Visfatin is a multifaceted molecule that exerts regulation effects on inflammation and apoptosis in RAW264.7 cells and mice immune organs.

Visfatin, a multifunctional adipocytokine, is particularly important in the regulation of apoptosis and inflammation through an unidentified mechanism. Clarifying the control mechanisms of visfatin on inflammation and apoptosis in RAW264.7 cells and mice immunological organs was the goal of the current investigation. In order to create a pathophysiological model, the RAW264.7 cells were stimulated with 200 ng/mL visfatin and 20 µg/mL lipopolysaccharide (LPS), either separately or combined. The effects of exogenous visfatin on inflammation and apoptosis in RAW264.7 cells were investigated by flow cytometry assay, RNA-seq analysis and fluorescence quantitative PCR. According to the findings, exogenous visfatin exhibits dual effects on inflammation by modulating the expression of IL-1α, TNFRSF1B, and LIF as well as taking part in various signaling pathways, including the MAPK and Rap1 signaling pathways. By controlling the expression levels of Bcl2l1, Bcl2a1a, and Fas and primarily participating in the PI3K/AKT signaling pathway and Hippo signaling pathway, exogenous visfatin can inhibit apoptosis in RAW264.7 cells. The visfatin inhibitor FK866 was used to further confirm the effects of visfatin on inflammation and apoptosis in mice immune organs. Subsequently, mice spleen and thymus were collected. It is interesting to note that in LPS-treated mice, suppression of endogenous visfatin might worsen the immune system's inflammatory response and even result in rapid mortality. Additionally, endogenous visfatin promotes the apoptosis in mice immune organs by regulating the expression levels of Bcl2l1, Fas, Caspase 3, Bcl2a1a, and Bax. Together, these results imply that visfatin is a multifaceted molecule that regulates inflammation and apoptosis in RAW264.7 cells and mice immunological organs by taking part in a variety of biological processes and regulating the amounts of associated cytokines expression. Our findings offer additional understandings of how visfatin affects apoptosis and inflammation.

CpxAR of Actinobacillus pleuropneumoniae Contributes to Heat Stress Response by Repressing Expression of Type IV Pilus Gene apfA.

Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.

The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, a major bacterial porcine respiratory tract pathogen causing pig pleuropneumonia, has resulted in high economic losses worldwide. The mutation of the two-component system CpxAR strongly impacted the virulence of A. pleuropneumoniae, but the underlying regulatory mechanism remained unclear. Here, we found that CpxAR positively regulated the cpxDCBA gene cluster involved in polysaccharide capsule export. A capsular layer was confirmed in wild-type cells by transmission electron microscopy, whereas cpxAR and cpxD mutants were non-capsulated. The mutants for polysaccharide capsule export gene cpxD exhibited non-capsulated and were strongly impaired in virulence for mice, indicating a major role of CPS export system in virulence. We then demonstrated that CpxR directly regulated the transcription of the CPS export gene cluster cpxDCBA. Taken together, our data suggested that CpxAR is a key modulator of capsule export that facilitates A. pleuropneumoniae survival in the host.

The VraSR two-component signal transduction system contributes to the damage of blood-brain barrier during Streptococcus suis meningitis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause high morbidity and mortality in both humans and swine. As the most important life-threatening infection of the central nervous system (CNS), meningitis is an important syndrome of S. suis infection. The vancomycin resistance associated sensor/regulator (VraSR) is a critical two-component signal transduction system that affects the ability of S. suis to resist the host innate immune system and promotes its ability to adhere to brain microvascular endothelial cells (BMECs). Prior work also found mice infected with ΔvraSR had no obvious neurological symptoms, unlike mice infected with wild-type SC19. Whether and how VraSR participates in the development of S. suis meningitis remains unknown. Here, we found ΔvraSR-infected mice did not show obvious meningitis, compared with wild-type SC19-infected mice. Moreover, the proinflammatory cytokines and chemokines in serum and brains of ΔvraSR-infected mice, including IL-6, TNF-α, MCP-1 and IFN-γ, were significantly lower than wild-type infected group. Besides, blood-brain barrier (BBB) permeability also confirmed that the mutant had lower ability to disrupt BBB. Furthermore, in vivo and in vitro experiments showed that SC19 could increase BBB permeability by downregulating tight junction (TJ) proteins such as ZO-1, ß-Catenin, Occludin, and Clauidn-5, compared with mutant ΔvraSR. These findings provide new insight into the influence of S. suis VraSR on BBB disruption during the pathogenic process of streptococcal meningitis, thereby offering potential targets for future preventative and therapeutic strategies against this disease.

The Metabolic Adaptation in Response to Nitrate Is Critical for Actinobacillus pleuropneumoniae Growth and Pathogenicity under the Regulation of NarQ/P.

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of ΔnarQ, the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP, and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

Mn uptake system affects the virulence of Streptococcus suis by mediating oxidative stress.

Manganese (Mn) is an important micronutrient that is not readily available to pathogens during infection. Hosts resist the invasion of pathogens through nutritional immunity and oxidative stress. To overcome this nutrient restriction, bacteria utilize high affinity transporters to compete with nutrient-binding proteins (e.g., calprotectin). Little is known about the role of Mn in the pathophysiology of Streptococcus suis. Here, we revealed that the tolerance of S. suis to calprotectin and oxidative stress was associated with Mn. Inactivation of Mn uptake system, TroABCD, in S. suis decreased the tolerance to calprotectin and oxidative stress. Furthermore, Mn uptake system mutant strains reduced capacity for bacterial cellular survival, and attenuated virulence in a mouse model. To explore the regulatory mechanism, we determined the transcriptional start site of troABCD using capping rapid amplification of cDNA ends. Furthermore, we revealed that TroR was a transcriptional regulatory repressor of troABCD. In the absence of troR, transcription levels of troA, troB, troC, and troD were not inhibited by low or high Mn levels, and intracellular Mn contents of mutant strains were higher than that of the wild-type strain. Finally, we used electrophoretic mobility shift assay to demonstrate that TroR bound the promoter region of troABCD. Collectively, this study revealed that Mn acquisition was essential for pathogenesis of S. suis and Mn uptake systems should be targets for the development of new antimicrobials.

Role of the Two-Component System CiaRH in the Regulation of Efflux Pump SatAB and Its Correlation with Fluoroquinolone Susceptibility.

Streptococcus suis is an important pathogen in both pigs and humans. Although the diseases associated with S. suis can typically be treated with antibiotics, such use has resulted in a sustained increase in drug resistance. Bacteria can sense and respond to antibiotics via two-component systems (TCSs). In this study, the TCS CiaRH was identified as playing an important role in the susceptibility of S. suis to fluoroquinolones (FQs). We found that a ΔciaRH mutant possessed lower susceptibility to FQs than the wild-type strain, with no observed growth defects at the tested concentrations and lower levels of intracellular drugs and dye. Proteomic data revealed that the levels of SatA and SatB expression were upregulated in the ΔciaRH mutant compared with their levels in the wild-type strain. The satA and satB genes encode a narrow-spectrum FQ efflux pump. The phenomena associated with combined ciaRH-and-satAB deletion mutations almost returned the ΔciaRH ΔsatAB mutant to the phenotype of the wild-type strain compared to the phenotype of the ΔciaRH mutant, suggesting that the resistance of the ΔciaRH strain to FQs could be attributed to satAB overexpression. Moreover, SatAB expression was regulated by CiaR (a response regulator of CiaRH) and SatR (a regulator of the MarR family). The ciaRH genes were consistently downregulated in response to antibiotic stress. The results of electrophoretic mobility shift assays (EMSAs) and affinity assays revealed that both regulator proteins directly controlled the ABC transporter proteins SatAB. Together, the results show that cascade-mediated regulation of antibiotic export by CiaRH is crucial for the ability of S. suis to adapt to conditions of antibiotic pressure. Our study may provide a new target for future antibiotic research and development. IMPORTANCE Streptococcus suis is a zoonotic pathogen with high incidence and mortality rates in both swine and humans. Following antibiotic treatment, the organism has evolved many resistance mechanisms, among which efflux pump overexpression can promote drug extrusion from the cell. This study clarified the role of CiaRH in fluoroquinolone resistance. A mutant with the ciaRH genes deleted showed decreased susceptibility to the antibiotics tested, an invariant growth rate, and reduced intracellular efflux pump substrates. This research also demonstrated that overexpression of the efflux pump SatAB was the main cause of ΔciaRH resistance. In addition, CiaR could combine with the promoter region of satAB to further directly suppress target gene transcription. Simultaneously, satAB was also directly regulated by SatR. Our findings may provide novel insights for the development of drug targets and help to exploit corresponding inhibitors to combat bacterial multidrug resistance.

PerR-Regulated Manganese Import Contributes to Oxidative Stress Defense in Streptococcus suis.

Streptococcus suis has been increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role in the antioxidative capability of bacteria, thus facilitating the escape of pathogenic species from the innate immunity systems of hosts. Here, we revealed that manganese increased the ability of S. suis to resist oxidative stress. RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular manganese homeostasis. Four genes, termed troABCD, were identified by NCBI BLASTp analysis. The troA, troB, troC, and troD deletion mutant strains exhibited decreased intracellular manganese content and tolerance to H2O2 compared to the wild-type strain. Thus, troABCD were determined to be involved in manganese uptake and played an important role in H2O2 tolerance in S. suis. Furthermore, the inactivation of perR increased the survival of H2O2-pulsed S. suis 2.18-fold and elevated the intracellular manganese content. H2O2-pulsed S. suis and perR deletion mutants upregulated troABCD. This finding suggested that H2O2 released the suppression of troABCD by perR. In addition, an electrophoretic mobility shift assay (EMSA) showed that PerR at 500 ng binds to the troABCD promoter, indicating that troABCD were directly regulated by PerR. In conclusion, this study revealed that manganese increases tolerance to H2O2 by upregulating the expression of troABCD. Moreover, PerR-regulated Mn import in S. suis and increased the tolerance of S. suis to oxidative stress by regulating troABCD. IMPORTANCE During infection, it is extremely important for bacteria to defend against oxidative stress. While manganese plays an important role in this process, its role is unclear in S. suis. Here, we demonstrated that manganese increased S. suis tolerance to oxidative stress. Four manganese ABC transporter genes, troABCD, were identified. Oxidative stress increased the content of manganese in the cell. Furthermore, PerR increased the tolerance to oxidative stress of S. suis by regulating troABCD. Manganese played an important role in bacterial defense against oxidative stress. These findings provide novel insight into the mechanism by which S. suis resists oxidative stress and approaches to inhibit bacterial infection by limiting manganese intake.

The CpxA/CpxR two-component system mediates regulation of Actinobacillus pleuropneumoniae cold growth.

Introduction: To survive in various hostile environments, two-component system is an adaptive mechanism for diverse bacteria. Activity of the CpxA/CpxR two-component system contributes to coping with different stimuli, such as pH, osmotic and heat stress. Methods: However, the role of the CpxA/CpxR system in cold resistance is little-known. In this study, we showed that CpxA/CpxRwas critical for A. pleuropneumoniae growth under cold stress. Results: ß-Galactosidaseanalysis showed that CpxA/CpxR positively regulated the predicted cold stress gene cspC. The mutant for cold stress gene cspC was impaired in the optimal growth of A. pleuropneumoniae under cold stress. Furthermore, electrophoretic mobility shift assays demonstrated that CpxR-P could directly regulate the transcription of the cold stress gene cspC. Discussion: These results presented in this study illustrated that the CpxA/CpxR system plays an important role in cold resistance by upregulating expression of CspC. The data give new insights into how A. pleuropneumoniae survives in cold stress.

Binding of Plasminogen to Streptococcus suis Protein Endopeptidase O Facilitates Evasion of Innate Immunity in Streptococcus suis.

The Gram-positive bacterial species Streptococcus suis is an important porcine and human pathogen that causes severe life-threatening diseases associated with high mortality rates. However, the mechanisms by which S. suis evades host innate immunity remain elusive, so identifying novel virulence factors involved in immune evasion is crucial to gain control over this threatening pathogen. Our previous work has shown that S. suis protein endopeptidase O (SsPepO) is a novel fibronectin-binding protein. Here, we identified that recombinant SsPepO binds human plasminogen in a dose-dependent manner. Moreover, the binding of SsPepO and plasminogen, upon the activation of urokinase-type plasminogen activator, generated plasmin, which could cleave complement C3b, thus playing an important role in complement control. Additionally, a SspepO-deficient mutant showed impaired adherence to plasminogen as well as impaired adherence to and invasion of rat brain microvascular endothelial cells compared with the wildtype strain. We further found that the SspepO-deficient mutant was efficiently killed by human serum and blood. We also confirmed that the SspepO-deficient mutant had a lower mortality rate than the wildtype strain in a mouse model. In conclusion, these results indicate that SsPepO is a novel plasminogen-binding protein that contributes to S. suis immune evasion.

CopA Protects Actinobacillus pleuropneumoniae against Copper Toxicity.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium causing porcine pleuropneumonia and severe economic losses in the global swine industry. The toxic trace element copper is required for many physiological and pathological processes in organisms. However, CopA, one of the most well-characterized P-type ATPases contributing to copper resistance, has not been characterized in A. pleuropneumoniae. We used quantitative PCR analysis to examine expression of the copA gene in A. pleuropneumoniae and investigated sequence conservation among serotypes and other Gram-negative bacteria. Growth characteristics were determined using growth curve analyses and spot dilution assays of the wild-type strain and a △copA mutant. We also used flame atomic absorption spectrophotometry to determine intracellular copper content and examined the virulence of the △copA mutant in a mouse model. The copA expression was induced by copper, and its nucleotide sequence was highly conserved among different serotypes of A. pleuropneumoniae. The amino acid sequence of CopA shared high identity with CopA sequences reported from several Gram-negative bacteria. Furthermore, the △copA mutant exhibited impaired growth and had higher intracellular copper content compared with the wild-type strain when supplemented with copper. The mouse model revealed that CopA had no influence on the virulence of A. pleuropneumoniae. In conclusion, these results demonstrated that CopA is required for resistance of A. pleuropneumoniae to copper and protects A. pleuropneumoniae against copper toxicity via copper efflux.