Characterization of a Unique ClpB Protein of Mycoplasma pneumoniae and Its Impact on Growth

Abstract

Mycoplasma pneumoniae accounts for 20 to 30% of all community-acquired pneumonia and has been associated with other airway pathologies, including asthma, and a range of extrapulmonary manifestations. Although the entire genomic sequence of M. pneumoniae has been completed, the functions of many of these genes in mycoplasma physiology are unknown. In this study, we focused on clpB, a well-known heat shock gene in other bacteria, to examine its role in mycoplasma growth. Transcriptional and translational analyses of heat shock in M. pneumoniae indicated that clpB is significantly upregulated, reinforcing its status as a critical responder to heat stress. Interestingly, M. pneumoniae ClpB does not use dual translational start points for ClpB synthesis, like other ClpB-characterized bacteria. Biochemical characterization of purified M. pneumoniae recombinant ClpB revealed casein-and lysine-independent ATPase activity and DnaK-DnaJ-GrpEdependent chaperone activity. An M. pneumoniae mini-Tn4001-integrated, clpB-null mutant was impaired in its ability to replicate under permissive growth conditions, demonstrating the growth-promoting status of ClpB.Mollicutes are unusual prokaryotes that lack bacterial cell walls and exhibit limited metabolic capabilities due to their streamlined genomes. Mycoplasma pneumoniae, with a genome of 816 kb, is confined to a parasitic life-style in humans (16). The importance of M. pneumoniae as a causative agent of acute and chronic respiratory diseases and extrapulmonary pathologies has been well documented by epidemiological studies in many settings. M. pneumoniae accounts for 20 to 30% of all community-acquired pneumonia and has been implicated in asthma and chronic obstructive pulmonary disease (3,36). This mycoplasma utilizes several attachment mechanisms to bind to sialylated and sulfated receptors on human target cells and to abundant host proteins, like fibronectin and surfactant protein A (2,7,18,19). An understanding of how M. pneumoniae responds to in vivo environmental signals and successfully establishes residence in the host has been elusive. This is compounded by the absence of well-established prokaryotic regulatory signaling, such as alternative sigma factors and twocomponent systems. For example, the presence of only one sigma factor (sigA, mpn352) in M. pneumoniae suggests that its response to external stimuli is not controlled by the level of expression of alternative sigma factors. However, the presence of the conserved inverted repeat sequence (CIRCE [controlling inverted repeat of chaperone expression]) immediately in front of four heat shock genes (dnaK, lonA, dnaJ, and clpB) and the identification of the CIRCE-associated repressor (hrcA, mpn124) provide evidence for negative regulation of gene expression in M. pneumoniae.Recent transcriptional studies of heat shock responses in M. pneumoniae (39), Mycoplasma genitalium (29), and Mycoplasma hyopneumoniae (6) have provided additional insights concerning the upregulation of specific heat shock genes. For example, in M....

Infection with Francisella tularensis LVSclpBLeads to an Altered yet Protective Immune Response

“…Due to ClpB's conserved nature and the finding that disruption of clpB attenuates F. tularensis as well as other bacteria (44)(45)(46)(47)(48), this gene is an excellent candidate target for attenuation of pathogenic bacteria for vaccine development. In the case of F. tularensis, LVS clpB infection was cleared faster than LVS yet induced a robust IFN-␥-mediated immune response that was protective in both short-and long-term secondary infections.…”

Inactivation of clpB in the Pathogen Leptospira interrogans Reduces Virulence and Resistance to Stress Conditions

“…A clpB mutant was discovered during a systematic search for insertional mutants obtained by random transposon mutagenesis in the pathogen Leptospira interrogans (24). In previous studies, clpB mutants of Salmonella enterica serovar Typhimurium, Mycoplasma pneumoniae, Porphyromonas gingivalis, Listeria monocytogenes, and Francisella tularensis have been shown to be relatively avirulent compared with their parental strains (5,6,15,22,38). In the present study, inactivation of the clpB gene of L. interrogans resulted in a mutant impaired in in vitro growth under stress conditions and exhibiting attenuation in virulence.…”

Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells

“…ClpB cooperates with the members of Hsp70 and Hsp40 chaperone families in resolubilization and reactivation of aggregated proteins that accumulate in cells under conditions of stress (Zolkiewski, Zhang and Nagy 2012;Mogk, Kummer and Bukau 2015). ClpB supports virulence and survival of a number of bacterial and protozoan pathogens including Staphylococcus aureus (Frees et al 2004), Pseudomonas aeruginosa (Lehoux, Sanschagrin and Levesque 2002), Salmonella typhimurium (Turner et al 1998), Shigella dysenteriae (Pieper et al 2009;Kuntumalla et al 2011), Porphyromonas gingivalis (Capestany et al 2008), Mycoplasma pneumoniae (Kannan et al 2008), Francisella tularensis (Meibom et al 2008), Enterococcus faecalis (de Oliveira et al 2011), Mycobacterium tuberculosis (Estorninho et al 2010;Vaubourgeix et al 2015), Leptospira interrogans (Lourdault et al 2011), Leischmania donovani (Krobitsch and Clos 1999) and Plasmodium falciparum (Beck et al 2014;Elsworth et al 2014). These established links between ClpB and the infectivity and survival of multiple different pathogens suggest that host-induced stress can result in misfolding and aggregation of pathogen's proteins and that their functional restoration can be mediated by ClpB.…”