Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo

Abstract

g Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle

“…Though its applicability to B . burgdorferi has been controversial (Wormser and Schwartz, ; Wormser et al ., ; Iyer et al ., ), persistence is a widely accepted phenomenon in microbiology which in some instances can have therapeutic implications (Dahl et al ., ; Lewis, ; Amato et al ., ; Zhang, ; Putrins et al ., ; Brauner et al ., ; Chuang et al ., ; Corrigan et al ., ). Persisters, while non‐dividing, appear to be metabolically active (Michiels et al ., ).…”

“…The term 'bacterial persistence' is used to describe the ability of pathogenic bacteria ('persisters') to survive in infected host tissues despite the presence of effective levels of antimicrobials and antibacterial cellular and humoral immunity (Lewis, 2010;Nguyen et al, 2011;Balaban et al, 2013;Conlon et al, 2015;Michiels et al, 2016). Though its applicability to B. burgdorferi has been controversial (Wormser and Schwartz, 2009;Wormser et al, 2012;Iyer et al, 2013), persistence is a widely accepted phenomenon in microbiology which in some instances can have therapeutic implications (Dahl et al, 2003;Lewis, 2010;Zhang, 2014;Putrins et al, 2015;Brauner et al, 2016;Chuang et al, 2016;Corrigan et al, 2016).…”

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid‐tolerance

“…Intracellular polyP levels are known to be important for maintaining susceptibility to INH (Thayil et al ., ; Chuang et al ., ; Wakamoto et al ., ; Chuang et al ., ) and some other anti‐tuberculosis drugs (Singh et al ., ). However, a recent study has shown that accumulation of polyP in mycobacteria increased susceptibility to β‐lactams and clofazimine (Chuang et al ., ). We do not know why polyP has different actions in controlling susceptibility to different drugs.…”

Antibiotic treatment shapes antigen presentation during chronic TB infection, offering novel targets for therapeutic vaccination

“…At day 0, 10 6 macrophages were infected with 5x10 6 of logarithmically growing H37Rv bacilli. The cells were harvested 4 days after infection, RNA was extracted and qPCR analysis was performed, as described previously [19]. The primers were listed in Table 1.…”

“…Although the Mtb stringent response contributes to the formation of persisters and antibiotic tolerance, it remains to be determined whether this pathway can be targeted therapeutically during chronic Mtb infection. We have shown previously that DNA vaccination with four key stringent response genes (relMtb, sigE, ppk2, and ppx1) generated antigen-specific CD4 + T-cell responses and vaccination prior to Mtb aerosol challenge augmented the tuberculocidal activity of isoniazid in mice [19]. However, very limited data are available regarding antigen presentation of stringent response factors during antitubercular treatment in vivo, as well as the optimal target for therapeutic DNA vaccination, which is an effective strategy for generating cellular and humoral immunity in various diseases [20].…”