Biological characteristics of manganese transporter MntP in Klebsiella pneumoniae.

Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety of infections. It is critical for bacteria to maintain metal homeostasis during infection. By using an isogenic mntP deletion mutant of K. pneumoniae strain NTUH-K2044, we found that MntP was a manganese efflux pump. Manganese increased the tolerance to oxidative stress, and oxidative stress could increase the intracellular manganese concentration. In oxidative stress, the mntP deletion mutant exhibited significantly higher sensitivity to manganese. Furthermore, iron could increase the tolerance of the mntP deletion mutant to manganese. Inductively coupled plasma mass spectrometry analysis revealed that the mntP deletion mutant had higher intracellular manganese and iron concentrations than wild-type and complementary strains. These findings suggested that iron could increase manganese tolerance in K. pneumoniae. This work elucidated the role of MntP in manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.IMPORTANCEMetal homeostasis plays an important role during the process of bacterial infection. Herein, we revealed that MntP was involved in intracellular manganese homeostasis. Manganese promoted resistance to oxidative stress in Klebsiella pneumoniae. Furthermore, we demonstrated that the mntP deletion mutant exhibited significantly lower survival under manganese and H2O2 conditions. Oxidative stress increased the intracellular manganese content of the mntP deletion mutant. MntP played a critical role in maintaining intracellular manganese and iron concentrations. MntP contributed to manganese detoxification and Mn/Fe homeostasis in K. pneumoniae.

DivIVA Interacts with the Cell Wall Hydrolase MltG To Regulate Peptidoglycan Synthesis in Streptococcus suis.

Bacterial morphology is largely determined by the spatial and temporal regulation of peptidoglycan (PG) biosynthesis. Ovococci possess a unique pattern of PG synthesis different from the well studied Bacillus, and the mechanism of the coordination of PG synthesis remains poorly understood. Several regulatory proteins have been identified to be involved in the regulation of ovococcal morphogenesis, among which DivIVA is an important one to regulate PG synthesis in streptococci, while its mechanism is largely unknown. Here, the zoonotic pathogen Streptococcus suis was used to investigate the regulation of DivIVA on PG synthesis. Fluorescent d-amino acid probing and 3D-structured illumination microscopy found that DivIVA deletion caused abortive peripheral PG synthesis, resulting in a decreased aspect ratio. The phosphorylation-depleted mutant (DivIVA3A) cells displayed a longer nascent PG and became longer, whereas the phosphorylation-mimicking mutant (DivIVA3E) cells showed a shorter nascent PG and became shorter, suggesting that DivIVA phosphorylation is involved in regulating peripheral PG synthesis. Several DivIVA-interacting proteins were identified, and the interaction was confirmed between DivIVA and MltG, a cell wall hydrolase essential for cell elongation. DivIVA did not affect the PG hydrolysis activity of MltG, while the phosphorylation state of DivIVA affected its interaction with MltG. MltG was mislocalized in the ΔdivIVA and DivIVA3E cells, and both ΔmltG and DivIVA3E cells formed significantly rounder cells, indicating an important role of DivIVA phosphorylation in regulating PG synthesis through MltG. These findings highlight the regulatory mechanism of PG synthesis and morphogenesis of ovococci. IMPORTANCE The peptidoglycan (PG) biosynthesis pathway provides a rich source of novel antimicrobial drug targets. However, bacterial PG synthesis and its regulation is a very complex process involving dozens of proteins. Moreover, unlike the well studied Bacillus, ovococci undergo unusual PG synthesis with unique mechanisms of coordination. DivIVA is an important regulator of PG synthesis in ovococci, while its exact role in regulating PG synthesis remains poorly understood. In this study, we determined the role of DivIVA in regulating lateral PG synthesis of Streptococcus suis and identified a critical interacting partner, MltG, in which DivIVA influenced the subcellular localizations of MltG through its phosphorylation. Our study characterizes the detailed role of DivIVA in regulating bacterial PG synthesis, which is very helpful for understanding the process of PG synthesis in streptococci.

Antimicrobial compounds from an FDA-approved drug library with activity against Streptococcus suis.

AIM: Antimicrobial resistance (AMR) has become a global concern. Developing novel antimicrobials is one of the most effective approaches in tackling AMR. Considering its relatively low cost and risk, drug repurposing has been proposed as a valuable approach for novel antimicrobial discovery. The aim of this study was to screen for antimicrobial compounds against Streptococcus suis, an important zoonotic bacterial pathogen, from an Food and Drug Administration (FDA)-approved drug library. METHODS AND RESULTS: In this study, we tested the antimicrobial activity of 1815 FDA-approved drugs against S. suis. Sixty-seven hits were obtained that showed a growth inhibition of more than 98%. After excluding already known antibiotics and antiseptics, 12 compounds were subjected to minimal inhibition concentration (MIC) assessment against S. suis. This showed that pralatrexate, daunorubicin (hydrochloride), teniposide, aclacinomycin A hydrochloride and floxuridine gave a relatively low MIC, ranging from 0.85 to 5.25 µg/ml. Apart from pralatrexate, the remaining four drugs could also inhibit the growth of antimicrobial-resistant S. suis. It was also demonstrated that these four drugs had better efficacy against Gram-positive bacteria than Gram-negative bacteria. Cytotoxicity assays showed that floxuridine and teniposide had a relatively high 50% cytotoxic concentration (CC50 ). The in vivo efficacy of floxuridine was analysed using a Galleria mellonella larvae infection model, and the results showed that floxuridine was effective in treating S. suis infection in vivo. CONCLUSIONS: Five compounds from the FDA-approved drug library showed high antimicrobial activity against S. suis, among which floxuridine displayed potent in vivo efficacy that is worth further development. SIGNIFICANCE AND IMPACT OF STUDY: Our study identified several antimicrobial compounds that are effective against S. suis, which provides a valuable starting point for further antimicrobial development.

Comparative Phenotypic, Proteomic, and Phosphoproteomic Analysis Reveals Different Roles of Serine/Threonine Phosphatase and Kinase in the Growth, Cell Division, and Pathogenicity of Streptococcus suis.

Eukaryote-like serine/threonine kinases (STKs) and cognate phosphatases (STPs) comprise an important regulatory system in many bacterial pathogens. The complexity of this regulatory system has not been fully understood due to the presence of multiple STKs/STPs in many bacteria and their multiple substrates involved in many different physiological and pathogenetic processes. Streptococci are the best materials for the study due to a single copy of the gene encoding STK and its cognate STP. Although several studies have been done to investigate the roles of STK and STP in zoonotic Streptococcus suis, respectively, few studies were performed on the coordinated regulatory roles of this system. In this study, we carried out a systemic study on STK/STP in S. suis by using a comparative phenotypic, proteomic, and phosphoproteomic analysis. Mouse infection assays revealed that STK played a much more important role in S. suis pathogenesis than STP. The ∆stk and ∆stp∆stk strains, but not ∆stp, showed severe growth retardation. Moreover, both ∆stp and ∆stk strains displayed defects in cell division, but they were abnormal in different ways. The comparative proteomics and phosphoproteomics revealed that deletion of stk or stp had a significant influence on protein expression. Interestingly, more virulence factors were found to be downregulated in ∆stk than ∆stp. In ∆stk strain, a substantial number of the proteins with a reduced phosphorylation level were involved in cell division, energy metabolism, and protein translation. However, only a few proteins showed increased phosphorylation in ∆stp, which also included some proteins related to cell division. Collectively, our results show that both STP and STK are critical regulatory proteins for S. suis and that STK seems to play more important roles in growth, cell division, and pathogenesis.