Indranil Chatterjee

Staphylococcus aureus ClpC Is Required for Stress Resistance, Aconitase Activity, Growth Recovery, and Death

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and long-term survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimicrobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the catabolization of acetate during the stationary phase. clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary- and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.

In Vivo Mutations of Thymidylate Synthase (Encoded by thyA) Are Responsible for Thymidine Dependency in Clinical Small-Colony Variants of Staphylococcus aureus

Trimethoprim-sulfamethoxazole (SXT)-resistant Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a cofactor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to previously published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, and nupC) was performed by quantitative reverse transcription-PCR. The previously published sequences of thyA and two normal clinical strains were highly conserved, while thyA of four normal strains and four SCVs had nonsynonymous point mutations. In 8/10 SCVs, deletions occurred, resulting in stop codons which were located in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics, and transcription patterns. In conclusion, we demonstrated that mutations of the thyA gene were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA protein is necessary and sufficient to change the SCV phenotype and behavior back to normal.

Thymidine-dependent Staphylococcus aureus small-colony variants are associated with extensive alterations in regulator and virulence gene expression profiles.

ABSTRACT Chronic airway infection is a hallmark of cystic fibrosis (CF) and many CF patients are infected persistently by Staphylococcus aureus. Thymidine-dependent trimethoprim-sulfamethoxazole (SXT)-resistant S. aureus small-colony variants (SCVs), often in combination with isogenic normal S. aureus phenotypes, are highly prevalent and persistent in airway secretions of CF patients due to long-term SXT therapy (B. Kahl, M. Herrmann, A. S. Everding, H. G. Koch, K. Becker, E. Harms, R. A. Proctor, and G. Peters, J. Infect. Dis. 177:1023-1029, 1998). In this report, SCVs were compared to normal S. aureus by transcription analysis of important regulator (sigB, sarA, and agr) and virulence (α-hemolysin, hla, and protein A, spa) genes. Growth curve analyses revealed longer doubling times and lower final densities for SCVs than for normal strains. sigB activity was measured by transcription analysis of the sigB target gene asp23. For nearly all SCVs, expression of all regulators was decreased as assessed by asp23 reverse transcription-PCR for sigB and Northern analysis for sarA and agr. These results are in agreement with diminished hla signals in all SCVs and increased spa signals in 5 of 10 SCVs compared to the isogenic normal S. aureus. Both supplementation of SCVs with thymidine and activation of the agr quorum-sensing system by the supernatant of the isogenic normal strain reversed transcription to almost normal levels. In conclusion, multiple changes in growth characteristics and in regulator and virulence gene expression render SCVs less virulent and allow them to survive in the hostile environment present in the airways of CF patients, thereby illustrating adaptation of the bacteria during long-term persistence.

Enhanced Post-Stationary-Phase Survival of a Clinical Thymidine-Dependent Small-Colony Variant of Staphylococcus aureus Results from Lack of a Functional Tricarboxylic Acid Cycle

The mechanisms underlying the persistence of the Staphylococcus aureus small-colony variant (SCV) are not fully elucidated. In this study, clinical thymidine-dependent SCVs displayed altered expression of citB, clpC, and arcA genes, reduced acetate catabolization, and enhanced survival. These results implicate the importance of changes in tricarboxylic acid cycle and acetic acid metabolism in SCV survival and persistence.

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.

Characterization of Clinical Enterococcus faecalis Small-Colony Variants

ABSTRACT In this report, we present a clinical case of chronic aortic valve endocarditis caused by Enterococcus faecalis small-colony variants (SCVs), with ensuing characterization of the SCV phenotype in comparison to the clonally related normal phenotype with respect to alterations in microscopic and ultrastructural morphology, growth behavior, and metabolic pathways. In contrast to the normal phenotype, light and electron microscopy of the Enterococcus SCVs demonstrated the presence of heterogeneous cells of different sizes with aberrant shapes. Furthermore, SCVs showed excessive production of an intercellular substance and alterations in cell division displayed by a thick, coarse cell wall and incomplete, branched, and multiple cross walls without obvious cell separation. In addition, empty “ghost” cells were visible. In growth experiments, SCVs displayed an extended lag phase with delayed entrance into the stationary phase. Interestingly, SCV cells growing under aerobic conditions did not attain the growth and viability of the normal phenotype or those of SCVs growing under microaerobic conditions, suggesting impaired growth behavior and enhanced vulnerability in the presence of oxygen. By metabolite analysis, SCVs failed to produce significant amounts of acetate or lactate under aerobic growth conditions but were able to produce lactate under microaerobic growth conditions, implicating the induction of a fermentative metabolism. In conclusion, the observed structural alterations and changes in the cellular growth and metabolic pathways facilitated the survival of Enterococcus SCVs under microaerobic conditions in vitro and thus presumably in vivo during endocarditis.

Senescence of staphylococci: using functional genomics to unravel the roles of ClpC ATPase during late stationary phase

Disease caused by Staphylococcus aureus frequently takes a chronic persistent course, and such infections are difficult to treat. S. aureus has developed various stress response systems allowing for coordinated expression of virulence factors and adaptation to environmental conditions. Clp ATPase/protease complexes for protein reactivation and degradation are highly conserved systems with a primary function in stress response. In various bacterial species, the role of Clp complexes has been associated with competence, cell wall synthesis, virulence and other physiologic properties. More recently, in S. aureus various Clp ATPases have been found to influence global regulator functions resulting in complex phenotypic changes. In this review, we briefly outline current knowledge including our own work with ClpC ATPase. We could highlight an important role of ClpC that allows for post-stationary regrowth and entry into the bacterial death phase through a functional tricarboxylic acid (TCA) cycle metabolism. We have concluded that ClpC may play a major regulatory role for long-term survival. Furthermore, using functional genomics data, we could extend the global characterization of the functions of ClpC in S. aureus with respect to late-phase phenomena such as S. aureus carbon metabolism, ion homeostasis, oxidative stress response, survival, and programmed cell death. These studies will thus help to further unravel the putative role of Clp ATPases in the chronic-persistent course of disease.

Staphylococcus aureus ClpC is involved in protection of carbon-metabolizing enzymes from carbonylation during stationary growth phase

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Among them, ClpC, belonging to the Hsp100/Clp ATPase family, seems to play an important role. For instance, we previously demonstrated that a functional clpC deletion resulted in enhanced survival in the late stationary phase (death phase period) compared to the parental S. aureus strain. However, the mechanisms for the enhanced survival of a S. aureus clpC mutant during the death phase period are still elusive. In Escherichia coli, among the factors that might lead to bacterial cell death during stationary phase, the amount of protein aggregates and/or oxidized proteins appears to be of major importance. Thus, in the present study, we have evaluated protein aggregates and carbonylated protein (as a marker of protein oxidation) contents both in the wild type and in an S. aureus clpC mutant during the exponential growth phase and the death phase. Whereas at all time points the tested clpC mutant exhibits the same amount of protein aggregates as the WT strain, the total amount of carbonylated proteins appears to be lower in the clpC mutant. Moreover, we observed that at the entrance of the death phase carbon-metabolizing enzymes [such as the TCA cycle enzymes Mqo2 (malate: quinone oxidoreductase) and FumC/CitG (fumarate hydratase)] albeit not the bulk proteins are carbonylated to a larger extent in the clpC mutant. Reduced activity of the TCA cycle due to specific carbonylation of these proteins will result in a decrease of endogenous oxidative stress which in turn might confer enhanced survival of the clpC mutant during the death phase period thus contributing to bacterial longevity and chronic infection.

Investigations to the Antibacterial Mechanism of Action of Kendomycin.

Purpose The emergence of bacteria that are resistant to many currently used drugs emphasizes the need to discover and develop new antibiotics that are effective against such multi-resistant strains. Kendomycin is a novel polyketide that has a unique quinone methide ansa structure and various biological properties. This compound exhibits strong antibacterial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Despite the promise of kendomycinin in several therapeutic areas, its mode of action has yet to be identified. Methods In this study, we used a multidisciplinary approach to gain insight into the antibacterial mechanism of this compound. Results The antibacterial activity of kendomycin appears to be bacteriostatic rather than bactericidal. Kendomycin inhibited the growth of the MRSA strain COL at a low concentration (MIC of 5 μg/mL). Proteomic analysis and gene transcription profiling of kendomycin-treated cells indicated that this compound affected the regulation of numerous proteins and genes involved in central metabolic pathways, such as the tricarboxylic acid (TCA) cycle (SdhA) and gluconeogenesis (PckA and GapB), cell wall biosynthesis and cell division (FtsA, FtsZ, and MurAA), capsule production (Cap5A and Cap5C), bacterial programmed cell death (LrgA and CidA), the cellular stress response (ClpB, ClpC, ClpP, GroEL, DnaK, and GrpE), and oxidative stress (AhpC and KatA). Electron microscopy revealed that kendomycin strongly affected septum formation during cell division. Most kendomycin-treated cells displayed incomplete septa with abnormal morphology. Conclusions Kendomycin might directly or indirectly affect the cell division machinery, protein stability, and programmed cell death in S. aureus. Additional studies are still needed to obtain deeper insight into the mode of action of kendomycin.