Physiologic Effects of Forced Down-Regulation of dnaK and groEL Expression in Streptococcus mutans

Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL 32610-0424, USA.
Journal of Bacteriology (Impact Factor: 2.81). 04/2007; 189(5):1582-8. DOI: 10.1128/JB.01655-06
Source: PubMed


Strains of Streptococcus mutans lacking DnaK or GroEL appear not to be isolable. To better distinguish the roles played by these chaperones/chaperonins in
the physiology of S. mutans, we created a knockdown strategy to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80%
in strain SM13. Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels
of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent strain, had a strong
tendency to aggregate in broth culture, and showed major changes in their proteomes. Compared with the wild-type strain, SM12
and SM13 had impaired biofilm-forming capacities when grown in the presence of glucose. The SM12 strain was impaired in its
capacity to grow at 44°C or at pH 5.0 and was more susceptible to H2O2, whereas SM13 behaved like the wild-type strain under these conditions. Phenotypical reversions were noted for both mutants
when cells were grown in continuous culture at a low pH, suggesting the occurrence of compensatory mutations. These results
demonstrate that DnaK and GroEL differentially affect the expression of key virulence traits, including biofilm formation
and acid tolerance, and support that these chaperones have evolved to accommodate unique roles in the context of this organism
and its niche.

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    • "It is remarkable that among the differentially expressed proteins between the mutant and the wild type strain, some have been previously characterized as involved in biofilm formation in X. citri or in other pathogenic bacteria. Such is the case of DNA-directed RNA polymerase subunit β [32], tryptophan synthase [43], GroEL [44,45], FadL [32,42,46] and several TBDTs [42,47]. Interestingly, high intracellular L-tryptophan concentration prevents biofilm formation and triggers degradation of mature biofilm in E. coli[43]. "
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    ABSTRACT: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.
    BMC Microbiology 04/2014; 14(1):96. DOI:10.1186/1471-2180-14-96 · 2.73 Impact Factor
    • "DnaK is central in the molecular chaperone complex that includes DnaK, DnaJ and GrpE. In addition to its role in protein folding and in protecting cells from stress, DnaK plays a central role in induction of capsule (Genevaux et al., 2001) and also in expression of genes related to pathogenicity and virulence (Hanawa et al., 2002; Lemos et al., 2007). It has been shown in mice to be an important surface streptococcal virulence factor and protective antigen (Lemos et al., 2000; Fluegge et al., 2004; Kim et al., 2006). "
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    ABSTRACT: Streptococcus equi subspecies zooepidemicus (Sz) is a tonsillar and mucosal commensal of healthy horses with the potential to cause opportunistic infections of the distal respiratory tract stressed by virus infection, transportation, training or high temperature. The invasive clone varies from horse to horse with little evidence of lateral transmission in the group. Tonsillar isolates are non-mucoid although primary isolates from opportunist lower respiratory tract infections may initially be mucoid. In this study, a novel stably mucoid Sz (SzNC) from a clonal epizootic of respiratory disease in horses in different parts of New Caledonia is described. SzNC (ST-307) was isolated in pure culture from transtracheal aspirates and as heavy growths from 80% of nasal swabs (n = 31). Only 4% of swabs from unaffected horses (n = 25) yielded colonies of Sz. A viral etiology was ruled out based on culture and early/late serum antibody screening. Evidence for clonality of SzNC included a mucoid colony phenotype, SzP and SzM sequences, and multilocus sequence typing. SzNC, with the exception of isolates at the end of the outbreak, was hyaluronidase positive. Its SzP protein was composed of an N2 terminal, and HV4 variable region motifs and 18 carboxy terminal PEPK repeats. Biotin labeling of surface proteins revealed DnaK and alanyl-tRNA synthetase (AlaS) on the surface of clonal isolates, but not on non-clonal non-mucoid Sz from horses in the epizootic or unrelated US isolates. Reactivity of these proteins and SzP with convalescent serum indicated expression during infection.
    The Veterinary Journal 04/2014; 200(1). DOI:10.1016/j.tvjl.2014.01.014 · 1.76 Impact Factor
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    • "Several proteomics reports associate the up-regulation of DnaK to biofilm formation. Among them, a dnaK knock-down mutant of Streptococcus mutans with reduced levels of DnaK (<95%) shows impaired biofilm-forming capacity [30], while DnaK expression was up-regulated in a Prevotella intermedia biofilm-forming strain when compared to a variant lacking biofilm formation [31]. "
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    ABSTRACT: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms. In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study. Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.
    BMC Microbiology 08/2013; 13(1):186. DOI:10.1186/1471-2180-13-186 · 2.73 Impact Factor
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