Comparison of three methods for generating the coccoid form of Helicobacter pylori and proteomic analysis.

BACKGROUND: Helicobacter pylori changes from spiral to coccoid depending on the host state, environmental factors, and surrounding microbial communities. The coccoid form of H. pylori still maintains its complete cellular structure, retains virulence genes, and thus plays a role in pathogenicity. To understand the coccoid form, it is crucial to establish the in vitro generation of the coccoid H. pylori. Although some conditions have been studied for the generation of the coccoid form, few studies have compared these conditions for coccoid generation. Here, we generated coccoid forms via three methods and compared the differences in morphology, viability, culturability, and protein expression. RESULTS: The coccoid H. pylori was generated in vitro via three methods: a starvation method, a method using amoxicillin, and a method using the culture supernatant of Streptococcus mitis. The morphology and viability of the cells were examined by fluorescence microscopy after staining with SYTO9 and propidium iodide. The culturability of H. pylori was examined by counting colony-forming units on chocolate agar plates. In the starvation group, no colonies formed after 7 days, but viable coccoids were continuously observed. In the amoxicillin-treated group, the culturability decreased rapidly after 12 h, and showed a viable but non culturable (VBNC) state after the third day. Most cells treated with S. mitis supernatant changed to coccoid forms after 7 days, but colonies were continuously formed, probably due to living spiral forms. We performed proteomics to analyse the differences in protein profiles between the spiral and coccoid forms and protein profiles among the coccoid forms generated by the three methods. CONCLUSION: Amoxicillin treatment changed H. pylori to VBNC cells faster than starvation. Treatment with the S. mitis supernatant prolonged the culturability of H. pylori, suggesting that the S. mitis supernatant may contain substances that support spiral form maintenance. Proteomic analysis revealed that the expression of proteins differed between the spiral form and coccoid form of H. pylori, and this variation was observed among the coccoid forms produced via three different methods. The proteins in the coccoid forms produced by the three methods differed from each other, but common proteins were also observed among them.

Specialized digestive mechanism for an insect-bacterium gut symbiosis.

In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis.

Trehalose Biosynthesis Gene otsA Protects against Stress in the Initial Infection Stage of Burkholderia-Bean Bug Symbiosis.

Trehalose, a nonreducing disaccharide, functions as a stress protectant in many organisms, including bacteria. In symbioses involving bacteria, the bacteria have to overcome various stressors to associate with their hosts; thus, trehalose biosynthesis may be important for symbiotic bacteria. Here, we investigated the role of trehalose biosynthesis in the Burkholderia-bean bug symbiosis. Expression levels of two trehalose biosynthesis genes, otsA and treS, were elevated in symbiotic Burkholderia insecticola cells, and hence mutant ΔotsA and ΔtreS strains were generated to examine the functions of these genes in symbiosis. An in vivo competition assay with the wild-type strain revealed that fewer ΔotsA cells, but not ΔtreS cells, colonized the host symbiotic organ, the M4 midgut, than wild-type cells. The ΔotsA strain was susceptible to osmotic pressure generated by high salt or high sucrose concentrations, suggesting that the reduced symbiotic competitiveness of the ΔotsA strain was due to the loss of stress resistance. We further demonstrated that fewer ΔotsA cells infected the M4 midgut initially but that fifth-instar nymphs exhibited similar symbiont population size as the wild-type strain. Together, these results demonstrated that the stress resistance role of otsA is important for B. insecticola to overcome the stresses it encounters during passage through the midgut regions to M4 in the initial infection stage but plays no role in resistance to stresses inside the M4 midgut in the persistent stage. IMPORTANCE Symbiotic bacteria have to overcome stressful conditions present in association with the host. In the Burkholderia-bean bug symbiosis, we speculated that a stress-resistant function of Burkholderia is important and that trehalose, known as a stress protectant, plays a role in the symbiotic association. Using otsA, the trehalose biosynthesis gene, and a mutant strain, we demonstrated that otsA confers Burkholderia with competitiveness when establishing a symbiotic association with bean bugs, especially playing a role in initial infection stage. In vitro assays revealed that otsA provides the resistance against osmotic stresses. Hemipteran insects, including bean bugs, feed on plant phloem sap, which may lead to high osmotic pressures in the midguts of hemipterans. Our results indicated that the stress-resistant role of otsA is important for Burkholderia to overcome the osmotic stresses present during the passage through midgut regions to reach the symbiotic organ.

Proteolytic Activity of DegP Is Required for the Burkholderia Symbiont To Persist in Its Host Bean Bug.

Symbiosis requires the adaptation of symbiotic bacteria to the host environment. Symbiotic factors for bacterial adaptation have been studied in various experimental models, including the Burkholderia-bean bug symbiosis model. Previously identified symbiotic factors of Burkholderia symbionts of bean bugs provided insight into the host environment being stressful to the symbionts. Because DegP, which functions as both a protease and a chaperone, supports bacterial growth under various stressful conditions, we hypothesized that DegP might be a novel symbiotic factor of Burkholderia symbionts in the symbiotic association with bean bugs. The expression level of degP was highly elevated in symbiotic Burkholderia cells in comparison with cultured cells. When the degP-deficient strain competed for symbiotic association against the wild-type strain, the ΔdegP strain showed no symbiotic competitiveness. In vivo monoinfection with the ΔdegP strain revealed a lower symbiont titer in the symbiotic organ than that of the wild-type strain, indicating that the ΔdegP strain failed to persist in the host. In in vitro assays, the ΔdegP strain showed susceptibility to heat and high-salt stressors and a decreased level of biofilm formation. To further determine the role of the proteolytic activity of DegP in symbiosis, we generated missense mutant DegPS248A exhibiting a defect in protease activity only. The ΔdegP strain complemented with degPS248A showed in vitro characteristics similar to those of the ΔdegP strain and failed to persist in the symbiotic organ. Together, the results of our study demonstrated that the proteolytic activity of DegP, which is involved in the stress resistance and biofilm formation of the Burkholderia symbiont, plays an essential role in symbiotic persistence in the host bean bug. IMPORTANCE Bacterial DegP has dual functions as a protease and a chaperone and supports bacterial growth under stressful conditions. In symbioses involving bacteria, bacterial symbionts encounter various stressors and may need functional DegP for symbiotic association with the host. Using the Burkholderia-bean bug symbiosis model, which is a useful model for identifying bacterial symbiotic factors, we demonstrated that DegP is indeed a symbiotic factor of Burkholderia persistence in its host bean bug. In vitro experiments to understand the symbiotic mechanisms of degP revealed that degP confers resistance to heat and high-salt stresses. In addition, degP supports biofilm formation, which is a previously identified persistence factor of the Burkholderia symbiont. Furthermore, using a missense mutation in a protease catalytic site of degP, we specifically elucidated that the proteolytic activity of degP plays essential roles in stress resistance, biofilm formation, and, thus, symbiotic persistence in the host bean bug.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Unravelling the physiological roles of mazEF toxin-antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase.

BACKGROUND: Curiosity on toxin-antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. METHODS: This study aims to find out the impact of the mazEF toxin-antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR-cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. RESULTS: For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. CONCLUSIONS: Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug.

Ftsh Sensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics by Degrading YpfP, a Lipoteichoic Acid Synthesis Enzyme.

In the Gram-positive pathogen Staphylococcus aureus, FtsH, a membrane-bound metalloprotease, plays a critical role in bacterial virulence and stress resistance. This protease is also known to sensitize methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics; however, the molecular mechanism is not known. Here, by the analysis of FtsH substrate mutants, we found that FtsH sensitizes MRSA specifically to ß-lactams by degrading YpfP, the enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). Both the overexpression of FtsH and the disruption of ypfP-sensitized MRSA to ß-lactams were observed. The knockout mutation in ftsH and ypfP increased the thickness of the cell wall. The ß-lactam sensitization coincided with the production of aberrantly large LTA molecules. The combination of three mutations in the rpoC, vraB, and SAUSA300_2133 genes blocked the ß-lactam-sensitizing effect of FtsH. Murine infection with the ypfP mutant could be treated by oxacillin, a ß-lactam antibiotic ineffective against MRSA; however, the effective concentration of oxacillin differed depending on the S. aureus strain. Our study demonstrated that the ß-lactam sensitizing effect of FtsH is due to its digestion of YpfP. It also suggests that the larger LTA molecules are responsible for the ß-lactam sensitization phenotype, and YpfP is a viable target for developing novel anti-MRSA drugs.