Branched fatty acids inhibit the biosynthesis of menaquinone in Helicobacter pylori

Abstract

Menaquinone (MK) is an essential compound because it is an obligatory component of the electron transfer pathway in microorganisms. In Escherichia coli, MK was shown to be derived from chorismate by eight enzymes, designated MenA–H.1,2 However, we have revealed that an alternative pathway (we named it the futalosine pathway; Figure 1)3–5 was operating in some microorganisms including Helicobacter pylori, which causes gastric carcinoma. As humans and some useful intestinal bacteria, such as lactobacilli, possess the classical pathway, and MK biosynthesis is essential for survival of microorganisms,4 the futalosine pathway is an attractive target for the development of specific anti-H. pylori drugs. In this study, we tried to obtain such compounds from metabolites produced by actinomycetes and fungi. To identify compounds that specifically inhibit the futalosin pathway, we developed a screening method. We previously showed that the MqnA–D genes in the futalosine pathway were essential for survival, as these gene-disrupted Streptomyces coelicolor strains required exogenously added MK for their growth. Therefore, a compound that inhibits the growth of S. coelicolor but does not inhibit its growth in the presence of MK would become a candidate. However, this assay method is laborious and the growth of S. coelicolor is too slow to screen a mass of samples. Therefore, we employed a paper disk-agar diffusion assay, which is based on the phenomenon that antibiotics will diffuse from a paper disk into an agar medium containing test organisms and form a growth-inhibitory zone. We used two kinds of Bacillus strains as test organisms. One was Bacillus subtilis and the other is B. halodurans C-125. By genome sequencing, the latter strain was shown to be quite similar to the former strain in terms of genome size, G+C content of genomic DNA and the physiological properties used for taxonomical identification.6 Moreover, the phylogenetic placement of B. halodurans C-125 based on 16S rDNA sequence analysis indicated that this organism is more closely related to B. subtilis than to other members of the genus Bacillus.6 For example, both strains showed similar MIC values against representative antibiotics except for clarithromycin (Table 1). The resistance to clarithromycin was probably caused by the presence of an ermD gene that encodes the ribosome-methylation enzyme in B. halodurans.7 However, judging from the genome database of these strains, B. subtilis and B. halodurans C-125 use the classical pathway and the futalosine pathway, respectively, for the biosynthesis of MK.6 These facts suggested that a compound inhibiting the biosynthesis of MK in the futalosine pathway specifically represses the growth of only B. halodurans C-12. Therefore, we first screened candidate compounds for their ability to specifically inhibit B. halodurans C-125 using a paper disk assay. We tested approximately 1800 culture broths (1000 actinomycetes broths and 800 fungi broths). Of these, approximately 300 culture broths (17%) formed growth-inhibitory zone against both B. subtilis and B. halodurans C-125. However, we found that two actinomycetes culture broths specifically inhibited the growth of B. halodurans C-125 (hit ratio, 0.1%) (Figure 2). Then we examined whether B. halodurans C-125 could recover from this inhibition when MK (0.1 mg ml 1) was added into the culture broth during liquid cultivation. The growth of B. halodurans C-125 was clearly inhibited in the presence of sample no. AF50404, but this inhibition was reversed by adding MK, even in the presence of sample no. AF50404. This result strongly suggested that sample no. AF50404 contained a compound that specifically inhibited the futalosine pathway. The other candidate (AF50573) also showed the same inhibitory phenotype as that of no. AF50404, but it gradually lost its activity, probably because of its instability. Therefore, we used sample no. AF50404 in further analyses.