Regulator LdhR and d -Lactate Dehydrogenase LdhA of Burkholderia multivorans Play Roles in Carbon Overflow and in Planktonic Cellular Aggregate Formation

Abstract

LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in Burkholderia. Here, we show that gene ldhR of B. multivorans encoding an LTTR is cotranscribed with ldhA encoding a D-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic ΔldhR mutant grown in medium with 2% D-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including D-lactate, which was absent from the ΔldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 m in size after 24 h in liquid cultures, in contrast to ΔldhR mutant aggregates that never grew more than 60 m. The overexpression of D-lactate dehydrogenase LdhA in the ΔldhR mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment.IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the Burkholderia cepacia complex bacteria, which cause progressive deterioration of lung function that, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome." Burkholderia pathogenesis is multifactorial as they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study, we demonstrate the importance of a D-lactate dehydrogenase and a regulator in regulating carbon overflow, cellular aggregates, and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia patho-

ScmR, a global regulator of gene expression, quorum sensing, pH homeostasis, and virulence inBurkholderia thailandensis

“…Additionally, our RNA-Seq analyses revealed that ScmR stimulates expression of the putative lactate dehydrogenase LdhA-encoding gene, which is directly adjacent to scmR , and transcribed in the same direction in several Burkholderia spp. (26, 37) ( Fig. S2A ).…”

“…Because lactate dehydrogenase, by reducing pyruvate to lactate, was suggested to affect pH (26), ScmR could also intervene in pH homeostasis through the activation of ldhA transcription. Indeed, Silva et al (37) demonstrated that the Burkholderia multivorans ATCC 17616 homologue, called LdhR, influences pH homeostasis by activating both expression of the ldhA gene and lactate production. Still, we noticed no difference in pH between cultures of the B. thailandensis E264 wild-type strain and the ldhA -mutant (Fig.…”

“…3A), suggesting that LdhA is not involved in the ScmR-dependent control of pH homeostasis in B. thailandensis E264. Other discrepancies between the B. thailandensis E264 ScmR and the B. multivorans ATCC 17616 LdhR homologues were reported, such as their negative and positive effects on the development of biofilm, respectively (37). These findings indicate that these two proteins could be functionally different.…”

“…While the pathogenicity of B. multivorans ATCC 17616 in the Galleria mellonella larvae model of infection is apparently not regulated by the ScmR homologue LdhR (37), the loss of ScmR in both B. thailandensis E264 and B. pseudomallei Bp82 resulted in hypervirulence toward the model host C. elegans (26, 34). In addition, Melanson et al (38) demonstrated that the rice pathogen B. glumae 336gr-1 ScmR homologue, called NtpR, is a negative regulator of toxoflavin production, which is considered the primary virulence factor of B. glumae , suggesting that similarly to the B. thailandensis E264 and B. pseudomallei Bp82 ScmR homologues, NtpR influences virulence.…”

Impairment of CFTR activity in cultured epithelial cells upregulates the expression and activity of LDH resulting in lactic acid hypersecretion

“…On the other hand, in addition to reduce the pH, the increased lactate may contribute to the development of infections by supplying a carbon source to bacteria [64]. Also, particularly in diabetic patients, the glucose metabolism in bacteria may be an additional source of lactate, contributing to a reduced pH environment and to the microfilm formation [64,65].…”