Nε-Lysine Acetylation of the Histone-Like Protein HBsu Regulates the Process of Sporulation and Affects the Resistance Properties of Bacillus subtilis Spores

Bacillus subtilis produces dormant, highly resistant endospores in response to extreme environmental stresses or starvation. These spores are capable of persisting in harsh environments for many years, even decades, without essential nutrients. Part of the reason that these spores can survive such extreme conditions is because their chromosomal DNA is well protected from environmental insults. The α/β-type small acid-soluble proteins (SASPs) coat the spore chromosome, which leads to condensation and protection from such insults. The histone-like protein HBsu has been implicated in the packaging of the spore chromosome and is believed to be important in modulating SASP-mediated alterations to the DNA, including supercoiling and stiffness. Previously, we demonstrated that HBsu is acetylated at seven lysine residues, and one physiological function of acetylation is to regulate chromosomal compaction. Here, we investigate if the process of sporulation or the resistance properties of mature spores are influenced by the acetylation state of HBsu. Using our collection of point mutations that mimic the acetylated and unacetylated forms of HBsu, we first determined if acetylation affects the process of sporulation, by determining the overall sporulation frequencies. We found that specific mutations led to decreases in sporulation frequency, suggesting that acetylation of HBsu at some sites, but not all, is required to regulate the process of sporulation. Next, we determined if the spores produced from the mutant strains were more susceptible to heat, ultraviolet (UV) radiation and formaldehyde exposure. We again found that altering acetylation at specific sites led to less resistance to these stresses, suggesting that proper HBsu acetylation is important for chromosomal packaging and protection in the mature spore. Interestingly, the specific acetylation patterns were different for the sporulation process and resistance properties of spores, which is consistent with the notion that a histone-like code exists in bacteria. We propose that specific acetylation patterns of HBsu are required to ensure proper chromosomal arrangement, packaging, and protection during the process of sporulation.

A possible mechanism of farnesol tolerance in C. albicans biofilms implemented by activating the PKC signalling pathway and stabilizing ROS levels

Introduction. Biofilms are the natural growth state for most microorganisms. C. albicans biofilms are composed of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and elongated hyphal cells) encased in an extracellular matrix. C. albicans biofilms are notorious for resistance to antimicrobial treatments, a property that might be determined by complex mechanisms. Exogenous farnesol exerts a certain antifungal activity against C. albicans with medical implications. Different from other microbes, C. albicans biofilms can tolerate exogenous farnesol at high concentration with some cells still surviving and even maintaining proliferation, but the mechanism is unclear. Hypothesis. The study hypothesizes that C. albicans resists farnesol by activating the PKC signalling pathway. Aim. The study aims to discuss the molecular mechanism of C. albicans resistance to farnesol. Methodology. The ROS levels, the genes and proteins of the PKC pathway were compared between the farnesol-tolerant and non-tolerant groups using ROS levels assay, q-RT PCR and Western blot, respectively. Further, the mutant strains (pkc1Δ/Δ and mkc1Δ/Δ) were constructed, then the survival rates and ROS levels of biofilms exposed to farnesol were compared between mutant and wild strains. The morphological changes were observed using TEM. Results. The survival rates of C. albicans biofilms decreased rapidly under the lower concentration of farnesol (P<0.05), and kept stable (P>0.05) as the concentration rose up to 200 µM. The gene expression of the PKC pathway increased, while ROS levels remained stable and even decreased in the farnesol-tolerant biofilms, compared with those in the farnesol-nontolerant biofilms after farnesol treatment (P<0.05); pkc1 and mkc1 were significantly upregulated by C. albicans during the development of biofilm tolerance to farnesol. The cell wall and cytoplasm of pkc1Δ/Δ and mkc1Δ/Δ were damaged, and the ROS level increased (P<0.05); meanwhile, the survival rate of biofilms decreased compared with that of wild-type strain under the same farnesol concentrations (P<0.05). ROS inhibitors reversed these changes in pkc1Δ/Δ and mkc1Δ/Δ when the mutant strains exposed to farnesol. Conclusion. C. albicans biofilms can tolerate high concentrations of farnesol by activating pkc1 and mkc1 of the PKC pathway and stabilizing ROS levels. The pkc1 and mkc1 are two key genes regulated by C. albicans in the process of biofilm tolerance to farnesol.

The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot

Background Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. Methods We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. Results P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. Conclusions P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Dynamic Zero-Covid strategy curtails mutagenesis and emergence of new variants of the SARS-CoV-2

Within the local outbreak period of SARS-CoV-2 Delta variant in Nanjing and Yangzhou, China, we analyzed the mutation process of the Delta variants in 520 cases, as well as the production, spread and elimination of new mutant strains under the non-pharmaceutical interventions (NPI) strategy. The investigation on distribution of COVID-19 cases and phylogenetic analysis of SARS-CoV-2 genome sequences attributed to tracking the transmission chains, transmission chains were terminated by the isolation of the COVID-19 patients and quarantine of close-contracts, suggesting the importance of NPI in prompting some mutations to disappear and stopping the transmission of new variants. Dynamic zero-Covid strategy has been implemented successfully to against the second-largest local epidemic caused by an imported COVID-19 case in China.

Broad and Long-lasting Immune Response against SARS-CoV-2 Omicron and Other Variants by PIKA-Adjuvanted Recombinant SARS-CoV-2 Spike (S) Protein Subunit Vaccine (YS-SC2-010)

Recently SARS-CoV-2 Omicron (B.1.1.529) variant was identified in South Africa with numerous mutations in spike protein, and numerous community infections have been reported and raised grave concern around the world. Some studies found that the neutralization effects of several licensed vaccines against Omicron were dramatically reduced, which significantly affected antibody mediated protection, especially for individuals whose immunization were completed after extended period. In this regard, we studied the persistence and neutralization activity toward mutant strains in animal serum immunized with PIKA-adjuvanted recombinant SARS-CoV-2 spike protein subunit vaccine (YS-SC2-010). Here we are reporting that animal serum collected at 596 days after immunization with YS-SC2-010 still retains high and persistent neutralizing activity against all the Variant of Concern (VOC) variants, including Omicron variant. Although it is a blessed event to achieve 20 months long neutralization against Omicron variant after immunization with YS-SC2-010, it was also founded that the neutralization effect of immune serum on Omicron decreased by 6.29 folds as compared to D614G, more significantly when compared with other mutant strains.

The effect of acquired triazole resistance on abiotic stress tolerance and virulence in Candida auris micro evolved strains

Recently, C. auris become one of the most prominent members of the genus Candida. Since its occurrence, several C. auris outbreaks have been reported worldwide. These outbreaks were associated with isolates displaying decreased susceptibility towards fluconazole, the first-line agent for prophylaxis. Fluconazole is the most frequently used antifungal drug to treat bloodstream Candida infections. The physiological effects of acquired antifungal resistance was investigated in this species using fluconazole, posaconazole and voriconazole resistant mutant strains generated by the in vitro microevolution method. Alterations in antifungal susceptibility and cross resistance were determined by the microdilution method, utilizing azoles (fluconazole, voriconazole, posaconazole), echinocandins (caspofungin, micafungin, anidulafungin) and a polyene (amphotericin B). Changes in the abiotic stress tolerance was examined by spotting assay, using osmotic stressors, cell wall perturbants and a membrane detergent. To evaluate the impact of the acquired resistance on sterol biosynthesis, ergosterol composition of all generated mutant strains were examined. A potential relationship between virulence and acquired antifungal resistance was also studied both in vitro and in vivo. Phagocytosis of the generated strains by J774.2 mouse macrophage-like cells was measured and analyzed by flow cytometry. In the murine infection model fungal burden of the triazole evolved strains was determined in spleen, kidney, liver and brain and compared to the fungal burden associated with the initial azole susceptible strain. Significant differences in virulence of the initial and the generated strains was observed suggesting a potential connection between the virulence and antifungal susceptibility of the emerging fungal pathogen, C. auris.

Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses

Background: IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC. Methods: We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC. Results: ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents. Conclusions: CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.

Spontaneous Phage Resistance in Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide. The emergence of multidrug-resistant pathogens has renewed the interest in the therapeutic use of bacteriophages (phages). However, a major concern for the successful implementation of phage therapy is the emergence of phage-resistant mutants. The understanding of the phage-host interactions, as well as underlying mechanisms of resistance, have shown to be essential for the development of a successful phage therapy. Here, we demonstrate that the strictly lytic Escherichia phage vB_EcoM-P10 rapidly selected for resistance in the APEC ST95 O1 strain AM621. Whole-genome sequence analysis of 109 spontaneous phage-resistant mutant strains revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of these, a single SNP was detected while two SNPs were identified in a total of nine strains. In total, 34 unique SNPs were detected. In 42 strains, including 18 strains with SNP(s), gene losses spanning 17 different genes were detected. Affected by genetic changes were genes known to be involved in phage resistance (outer membrane protein A, lipopolysaccharide-, O- antigen-, or cell wall-related genes) as well as genes not previously linked to phage resistance, including two hypothetical genes. In several strains, we did not detect any genetic changes. Infecting phages were not able to overcome the phage resistance in host strains. However, interestingly the initial infection was shown to have a great fitness cost for several mutant strains, with up to ∼65% decrease in overall growth. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in APEC. Although acquired resistance to phages is frequently observed in pathogenic E. coli, it may be associated with loss of fitness, which could be exploited in phage therapy.

Estimating the transmission advantage of the D614G mutant strain of SARS-CoV-2, December 2019 to June 2020

Introduction The SARS-CoV-2 lineages carrying the amino acid change D614G have become the dominant variants in the global COVID-19 pandemic. By June 2021, all the emerging variants of concern carried the D614G mutation. The rapid spread of the G614 mutant suggests that it may have a transmission advantage over the D614 wildtype. Aim Our objective was to estimate the transmission advantage of D614G by integrating phylogenetic and epidemiological analysis. Methods We assume that the mutation D614G was the only site of interest which characterised the two cocirculating virus strains by June 2020, but their differential transmissibility might be attributable to a combination of D614G and other mutations. We define the fitness of G614 as the ratio of the basic reproduction number of the strain with G614 to the strain with D614 and applied an epidemiological framework for fitness inference to analyse SARS-CoV-2 surveillance and sequence data. Results Using this framework, we estimated that the G614 mutant is 31% (95% credible interval: 28–34) more transmissible than the D614 wildtype. Therefore, interventions that were previously effective in containing or mitigating the D614 wildtype (e.g. in China, Vietnam and Thailand) may be less effective against the G614 mutant. Conclusion Our framework can be readily integrated into current SARS-CoV-2 surveillance to monitor the emergence and fitness of mutant strains such that pandemic surveillance, disease control and development of treatment and vaccines can be adjusted dynamically.