TL;DR
It is proposed that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1, and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.
ABSTRACT ClpX functions as a component of the ClpXP protease, a conserved intracellular protease that regulates protein turnover, stress responses, and virulence in multiple bacterial species. Our lab has established that clpX is necessary for resistance to cell envelope targeting antibiotics, such as penicillin and daptomycin in Bacillus anthracis Sterne. Previous microarray data identified the msrA/B gene encoding a bifunctional methionine sulfoxide reductase as upregulated in the ΔclpX mutant. Methionine sulfoxide reductases (Msr) repair oxidatively damaged proteins by reducing methionine sulfoxide residues back to methionine. While Msr enzymes are primarily associated with oxidative stress, cell wall antibiotics induce expression of msrA1 and msrB in S. aureus. Here, we investigated the role of MsrA/B in oxidative and cell envelope stress. Our results show that although hydrogen peroxide and paraquat induce msrA/B expression, the ΔmsrA/B strain was not susceptible to either oxidant, whereas the ΔclpX strain was sensitive to both. We also found that loss of msrA/B conferred penicillin‐specific sensitivity, but, unlike ΔclpX, increased sensitivity was not seen with other cell wall or cell membrane targeting antibiotics. Inactivation of the catalytic cysteine of either Msr domain of MsrA/B failed to complement, suggesting that the reducing activity of MsrA/B is required for penicillin resistance. These findings indicate that while MsrA/B contributes to penicillin resistance, other proteins in the ClpXP modulon must also play a role in oxidative and cell envelope stress.
ABSTRACT The commensal and pathogenic lifestyles of the opportunistic fungal pathogen Candida albicans require complex signaling networks regulated by protein kinases. To investigate the role of C. albicans protein kinases at the intestinal epithelial interface, we screened a comprehensive protein kinase deletion library for the capacity of the mutants to damage intestinal epithelial cells (IEC). Mutants showing altered IEC cytotoxicity relative to the wild type were further analyzed for their growth and morphology, focusing on hyper-damaging strains to identify kinases that rather prevent host cell damage. Deletion of CRK1 caused increased IEC-specific damage, despite slower growth, reduced hyphal length, and reduced adhesion as compared to wild-type cells. While tissue invasion levels and the formation of transcellular tunnels of the crk1Δ/Δ mutant were increased, the translocation capacity through the IEC barrier was reduced. Transcriptional and metabolic profiling suggested a role for Crk1 in metabolic adaptation to carbon and nitrogen sources, which was validated by showing that high glucose and amino acids are required for crk1Δ/Δ to cause increased IEC damage. Deletion of CRK1 rendered C. albicans more susceptible to cell wall and membrane stressors, but caused higher resistance to a catalase-specific and histidine biosynthesis inhibitor. This phenotypic pattern of medium- and epithelial cell type-specific cytotoxicity displayed by a C. albicans protein kinase mutant suggests that Crk1 regulates processes linked to carbon and amino acid metabolism that are relevant to interactions with intestinal epithelial cells. IMPORTANCE Microbial signal transduction pathways regulate adaptation to changing environmental conditions and facilitate the success of many microbes during interactions with their hosts. The fungal pathobiont Candida albicans exists as a harmless commensal on mucosal surfaces of most humans but can also cause superficial and invasive infections under certain circumstances. Both lifestyles require complex signaling networks, predominantly regulated by protein kinases. The C. albicans genome was predicted to encode 108 protein kinases, yet nearly 50% remain uncharacterized. We aimed to dissect the role of C. albicans protein kinases during the transition from commensal to pathogen. We showed that multiple protein kinase genes are involved in epithelial cell damage. Particularly, the protein kinase gene Crk1 was of interest because deletion of CRK1 caused increased damage to intestinal epithelial cells under distinct conditions. Our study links Crk1 with regulation of metabolic processes relevant for commensalism and pathogenicity of C. albicans. Microbial signal transduction pathways regulate adaptation to changing environmental conditions and facilitate the success of many microbes during interactions with their hosts. The fungal pathobiont Candida albicans exists as a harmless commensal on mucosal surfaces of most humans but can also cause superficial and invasive infections under certain circumstances. Both lifestyles require complex signaling networks, predominantly regulated by protein kinases. The C. albicans genome was predicted to encode 108 protein kinases, yet nearly 50% remain uncharacterized. We aimed to dissect the role of C. albicans protein kinases during the transition from commensal to pathogen. We showed that multiple protein kinase genes are involved in epithelial cell damage. Particularly, the protein kinase gene Crk1 was of interest because deletion of CRK1 caused increased damage to intestinal epithelial cells under distinct conditions. Our study links Crk1 with regulation of metabolic processes relevant for commensalism and pathogenicity of C. albicans.
Findings, which suggest a mechanism that transiently halts cell separation when cell wall integrity is compromised, highlight a link between cell wall homeostasis and the regulation of cell division in yeast.
MKK2 mediates the phosphorylation of the Mkc1 MAPK in response to cell wall assembly interfering agents such as zymolyase or tunicamycin and also to oxidative stress and it is shown here that Mkk2 mediates the phosphorylation of the Mkc1 MAPK under different stress conditions.
A physical interaction is identified between AaSlt2 and Swi6/RlmA, suggesting that these components are critical for cell wall synthesis, which advances the understanding of pathogenic mechanisms of A. alternata and proposes potential strategies for controlling postharvest diseases.
Candida albicans is the most common invasive human fungal pathogen. We show that C. albicans thioredoxin reductase, Trr1, is an attractive antifungal target: it is essential at human body temperature, and fungal and human thioredoxin reductases are structurally divergent, predicting high selectivity of fungal-targeted inhibitors. TRR1 depletion directly impairs oxidative damage repair, but also triggers cascading disruption of stress signaling and metabolic adaptation. Impaired oxidative stress endurance and consequent amphotericin hypersensitivity are anticipated effects of TRR1 depletion. We unexpectedly find it also sensitizes Candida to cell wall stress and to a first-line echinocandin antifungal agent. TRR1-depleted cells have decreased cell wall glucan content. Driven by demand for NADPH reducing equivalents, these cells increase glucose-6-phosphate flux into the pentose phosphate pathway (PPP) as evinced by sharply elevated activity of the PPP’s first, rate-limiting enzyme. Since UDP-glucose—the substrate for cell wall glucan biosynthesis—is also derived from glucose-6-phosphate, we propose that metabolic pathway competition for this shared intermediate between NADPH production and cell wall glucan biosynthesis underlies the cell wall weakness of TRR1-depleted cells. Decreased activity of a key UDP-glucose biosynthetic enzyme supports this mechanism. Trr1 loss of function further drives feed-forward damage cycles: it accelerates respiration which increases reactive oxygen species, reduces gluconeogenesis which further limits glucose-6-phosphate availability, and suppresses oxidative- and cell wall stress signaling pathways. Our findings support Trr1 inhibition as a promising approach to improved treatment of C. albicans infections.
