Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation,Natural Transformation,and Antimicrobial Resistance

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.Campylobacter jejuni, a gram-negative, microaerophilic bacterium, occurs as a commensal among the intestinal microflora of various animals, especially chickens and cattle (6, 73). However, C. jejuni can infect human hosts, invading the intestinal mucosa and causing watery and/or bloody diarrhea (9). C. jejuni is transmitted to humans primarily through the consumption of contaminated chicken products, raw milk, or water (2, 3). Currently, C. jejuni is considered a leading bacterial cause of human food-borne gastroenteritis (3, 61) and has also been associated with a plethora of symptoms, including acute neuromuscular paralysis (Guillain-Barré syndrome) (26). Since an appropriate vaccine for human campylobacteriosis has yet to be introduced, it has been suggested that C. jejuni infections might be alternatively controlled by reducing colonization in food animals (73). Consequently, determining the physiological and genetic properties that allow the survival of C. jejuni and its colonization of animal hosts, pathogenicity, and adaptation to various stresses is of critical importance.The mechanisms underlying C. jejuni adaptation and survival under stresses imposed by its environment and host are not well understood. High variability between different C. jejuni strains and the unavailability of appropriate genetic tools and animal models have contributed to the lack of knowledge regarding its stress tolerance and pathogenicity. However, it is suggested that the capacity of C. jejuni to form viable-but-nonculturable (VBNC) cells under stress (14) and its readiness for natural transformation (68) and acquiring resistance to antibiotics (39) are among the strategies that promote stress adaptation and survival. Although little is known about the genetics underlying these processes, recent advances in C. jejuni genomics show that this bacterium carries several important genes that might play key roles in mediating stress adaptation and survival. Of particular interest are genes encoding polyphosphate (poly-P) kinases, ppk1 (CJJ81176_1361) and ppk2 (CJJ81176_0633), that were predicted to be involved in the metabolism of poly-P (22, 25, 47), an intracellular granule that impacts several physiological properties in many bacterial species, including pathogenicity, host colonization, adaptation to different environments, and survival (28, 31, 46).Poly-P kinase 1 (PPK1) is encoded by ppk1, which mediates the synthesis of all or most of the poly-P in the cell (33), while ppk2 encodes an enzyme (PPK2) that synthesizes GTP from poly-P (27). Both ppk genes have been associated with the metabolism of poly-P, which consists of phosphate residues that are linked by high-energy phosphoanhydride bonds and is widely distributed in bacterial species (60). Previous reports showed that poly-P plays important roles in bacterial survival and stress tolerance, including ATP production (8), entry of DNA through membrane channels (13, 54), capsule composition (67), maintaining nutritional requirements during starvation (34), motility, biofilm formation, and resistance to oxidative, osmotic, heat, acid and alkaline stresses, and stationary-phase survival (28, 31, 46, 48, 50, 52, 65). Because of their importance in many bacterial species, it is not surprising to assume a role for PPK and poly-P in C. jejuni survival, colonization, and stress tolerance (8).Interestingly, PPK1 has been shown to be important for C. jejuni stress responses and pathogenicity (10). However, the role of ppk1 in key metabolic and physiological responses of C. jejuni still needs further analysis. For instance, it has been proposed that during starvation, poly-P might act as a reservoir for phosphorus and energy (7). Subsequently, poly-P would be crucial for maintaining viability/metabolism in stressed cells. This has been observed in H. pylori, where the occurrence of poly-P correlated with culturability and structurally intact cells (45). Poly-P-containing nonculturable H. pylori showed a capacity for ATP and mRNA synthesis after a nutrient stimulus (45). Consequently, poly-P might be an important factor for the formation of VBNC cells by stressed bacteria, including C. jejuni. Furthermore, natural transformation is perhaps one of the most important mechanisms in the adaptation of C. jejuni, and poly-P has been reported to play a role in the entry of DNA through membrane channels (13, 54). It follows that poly-P might be important for natural transformation, adaptation, and acquisition of antibiotic resistance genes in C. jejuni. Poly-P can further impact the survival and adaptation in C. jejuni by modulating antibiotic resistance properties. For example, poly-P interacted with Escherichia coli ribosomes (42), which are known targets of several antibiotics. These observations suggest that ppk1 might be linked to important physiology and functions such as VBNC cell formation, natural transformation, and antimicrobial resistance in C. jejuni. Therefore, in the present study, we determined the contribution of PPK1 to C. jejuni stress responses and adaptation, including the ability to form VBNC cells under acid stress, natural transformation, and antimicrobial resistance. Furthermore, we assessed the impact of ppk1 deletion on in vivo chicken colonization. Our findings highlight the importance of PPK1 in C. jejuni survival, adaptation to different environmental stresses, and in vivo colonization. These findings also indicate the suitability of PPK1 as a potential target for controlling the proliferation of this pathogen.