Characterization of L-serine deaminases, SdaA (PA2448) and SdaB (PA5379), and their potential role inPseudomonas aeruginosapathogenesis

ABSTRACTRegardless of the site of infectivity, all pathogens require high energetic influxes. This energy is required to counterattack the host immune system and in the absence the bacterial infections are easily cleared by the immune system. This study is an investigation into one highly bioenergetic pathway inPseudomonas aeruginosainvolving the amino acid L-serine and the enzyme L-serine deaminase (L-SD).P. aeruginosais an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. L-SD has been linked directly to the pathogenicity of several organisms including but not limited toCampylobacter jejuni, Mycobacterium bovis,Streptococcus pyogenes, andYersinia pestis. We hypothesized thatP. aeruginosaL-SD is likely to be critical for its virulence. The genome sequence analysis revealed the presence of two L-SD homologs encoded bysdaAandsdaB.We analyzed the ability ofP. aeruginosato utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains ofsdaA(PAOsdaA) andsdaB(PAOsdaB) with their isogenic parentP. aeruginosaPAO1. We demonstrate thatP. aeruginosais unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine. Both SdaA and SdaB contribute to L-serine deamination, 34 % and 66 %, respectively. Glycine was also shown to increase the L-SD activity especially from SdaB. Glycine-dependent induction requires the inducer serine. The L-SD activity from both SdaA and SdaB is inhibited by the amino acid L-leucine. These results suggest thatP. aeruginosaL-SD is quite different from the characterizedE. coliL-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as sole carbon source were isolated. In addition, suicide vectors were constructed which allow for selective mutation of thesdaAandsdaBgenes on anyP. aeruginosastrain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

Detoxification of endogenous serine prevents cell lysis upon glucose depletion in bacteria

The amino acid serine, despite its diverse metabolic roles, can become toxic when present in excess. Indeed, many bacteria rapidly deaminate exogenously supplied serine into pyruvate and ammonia, even at the expense of biomass production. Here we report a surprising case in which endogenously produced serine must be detoxified in order for the bacteriumEscherichia colito survive. Specifically, we show thatE. colicells lacking thesdaCBoperon, which encodes a serine transporter and a serine deaminase, lyse upon glucose depletion when serine is absent from the growth medium. Lysis can be prevented by omission of glycine or by inhibition of the glycine cleavage system, suggesting that activation of glycine catabolism upon glucose depletion causes a transient increase in intracellular serine levels. Heterologous expression of the serine transporter SdaC is sufficient to prevent lysis, indicating a dominant role for serine export, rather than deamination, in mitigating serine toxicity. Since lysis can be modulated by altering alanine availability, we further propose that mis-incorporation of serine instead of alanine into peptidoglycan crosslinks is the cause of lysis. Together, our results reveal that SdaC-mediated detoxification of intracellularly produced serine plays a protective role during sudden shifts in nutrient availability in bacteria.Author summaryThe amino acid serine is a building block used to make many types of macromolecules, yet bacteria actively degrade serine that is provided in growth media. Serine degradation is thought to prevent toxic serine accumulation, but the biological role of this process is not fully understood. We observed that cells lacking thesdaCBoperon, which encodes a serine transporter and an enzyme that converts serine to pyruvate, suddenly lyse upon depletion of glucose from the growth medium. This surprising phenotype occurs only in media lacking serine, suggesting thatsdaCBis required to detoxify intracellularly produced serine. Expression of the serine transporter SdaC is sufficient to prevent lysis, providing the first evidence that serine export can be an essential function of this protein. Our results reveal that sudden shifts in nutrient availability can increase the intracellular concentration of useful metabolites to toxic levels and suggest that increasing intracellular serine levels by manipulating SdaC activity may be a possible antimicrobial strategy.

Significance of the d-Serine-Deaminase and d-Serine Metabolism of Staphylococcus saprophyticus for Virulence

ABSTRACTStaphylococcus saprophyticusis the only species ofStaphylococcusthat is typically uropathogenic and possesses a gene coding for ad-serine-deaminase (DsdA). Asd-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that thed-serine-deaminase gene is found in the genome of uropathogens. It has been suggested thatd-serine-deaminase or the ability to respond to or to metabolized-serine is important for virulence. For uropathogenicEscherichia coli(UPEC), a high intracellulard-serine concentration affects expression of virulence factors.S. saprophyticusis able to grow in the presence of highd-serine concentrations; however, itsd-serine metabolism has not been described. The activity of thed-serine-deaminase was verified by analyzing the formation of pyruvate fromd-serine in different strains with and withoutd-serine-deaminase. Cocultivation experiments were performed to show thatd-serine-deaminase confers a growth advantage toS. saprophyticusin the presence ofd-serine. Furthermore,in vivocoinfection experiments showed a disadvantage for the ΔdsdAmutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence ofd-serine. In addition, we show thatS. saprophyticusis able to used-serine as the sole carbon source, but interestingly,d-serine had a negative effect on growth when glucose was also present. Taken together,d-serine metabolism is associated with virulence inS. saprophyticus, as at least one known virulence factor is upregulated in the presence ofd-serine and a ΔdsdAmutant was attenuated in virulence murine model of urinary tract infection.