Lactate dehydrogenase A promotes communication between carbohydrate catabolism and virulence in Bacillus cereus

Abstract

The diarrheal potential of a Bacillus cereus strain is essentially dictated by the amount of secreted nonhemolytic enterotoxin (Nhe). Expression of genes encoding Nhe is regulated by several factors, including the metabolic state of the cells. To identify metabolic sensors that could promote communication between central metabolism and nhe expression, we compared four strains of the B. cereus group in terms of metabolic and nhe expression capacities. We performed growth performance measurements, metabolite analysis, and mRNA measurements of strains F4430/73, F4810/72, F837/76, and PA cultured under anoxic and fully oxic conditions. The results showed that expression levels of nhe and ldhA, which encodes lactate dehydrogenase A (LdhA), were correlated in both aerobically and anaerobically grown cells. We examined the role of LdhA in the F4430/73 strain by constructing an ldhA mutant. The ldhA mutation was more deleterious to anaerobically grown cells than to aerobically grown cells, causing growth limitation and strong deregulation of key fermentative genes. More importantly, the ldhA mutation downregulated enterotoxin gene expression under both anaerobiosis and aerobiosis, with a more pronounced effect under anaerobiosis. Therefore, LdhA was found to exert a major control on both fermentative growth and enterotoxin expression, and it is concluded that there is a direct link between fermentative metabolism and virulence in B. cereus. The data presented also provide evidence that LdhA-dependent regulation of enterotoxin gene expression is oxygen independent. This study is the first report to describe a role of a fermentative enzyme in virulence in B. cereus.

FIG. 1.

Metabolic responses of B. cereus strains F4430/73, F4810/72, F837/76, and PA. (A) Pathways for the excretion of partially oxidized metabolites. The reactions are represented by the names of the corresponding genes, as follows: ldhA, ldhB, and ldhC, l-lactate dehydrogenase; pfl, pyruvate formate lyase; pdh, pyruvate dehydrogenase; pta, phosphotransacetylase; ack, acetate kinase; adhE, acetaldehyde dehydrogenase; adhA, alcohol dehydrogenase; mdh, malate dehydrogenase; sdh, succinate dehydrogenase; citB, aconitase hydratase; citZ, citrate synthase. Reactions that normally function during aerobiosis are represented by gray arrows, and reactions that normally function during anaerobiosis are represented by black arrows. CIT, citrate; ICT, isocitrate; AKG, α-ketoglutarate; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; TCA, tricarboxylic acid cycle. (B) Extracellular metabolite analyses. (C) Specific Nhe production. Error bars indicate the standard errors of the means.

FIG. 2.

Operon mapping and expression of the B. cereus F4430/73 ldhA-pfo operon. (A) Large arrows represent the ldhA and pfo ORFs. The small arrowheads flag the positions and directions of primers used in RT-PCR. Lengths of expected PCR products are also shown for every primer pair. (B) RT-PCR analyses for detecting ldhA, pfo, and the intergenic (IG) region between ldhA and pfo in strain F4430/73. M, molecular mass marker. (C) The nucleotide sequence of the promoter region of F4430/73 ldhA is shown. The transcriptional start site (+1) and the putative −10 sequence are in bold and underlined. Putative regulatory sequences are highlighted in gray, the start codon is boxed, and inverted repeat sequences are indicated by arrows.

FIG. 3.

Cell-free expression of ldhA, ldhB, and ldhC. Lactate dehydrogenases were expressed as His-tagged proteins by using the RTS 500 E. coli HY kit in the presence of GroE supplement. Purified LdhA, LdhB, and LdhC were visualized by Western blotting using anti-His antibodies. (A) Purified LdhA (34.6 kDa). (B) Lane 1, purified LdhB (34.5 kDa); lane 2, purified LdhC (34.7 kDa). The sizes of the molecular mass markers (in kDa) are indicated on the left.

FIG. 4.

Amino acid sequence alignment of the three B. cereus l-lactate dehydrogenases. Black and gray boxes correspond to identical and similar amino acid residues, respectively. The tyrosine residue reported as acting in both dehydrogenase activity and ssDNA binding in eukaryotic cells is indicated with an asterisk (40). Dashes indicate gaps.