Role of capsular polysaccharide (<scp>CPS</scp>) in biofilm formation and regulation of <scp>CPS</scp> production by quorum‐sensing in <i><scp>V</scp>ibrio vulnificus</i>

Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance.IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.KEYWORDS lipopeptide, pumilacidin, Vibrio alginolyticus, motility, antibiotic, antimicrobial, antibiofilm B acterial motility is one of several crucial factors during the initial infection and colonization processes of pathogens. In particular, it helps pathogens overcome the repulsive forces between the bacterial cell wall and the host tissues and facilitates host attachment (1-3). The flagellum is one of the main locomotive organelles respon-

“…Cell aggregation is correlated to biofilm formation (32). Next, we investigated cell aggregation in the bacteria with or without biofilm formation capacity.…”

Section: Resultsmentioning

confidence: 99%

“…The growth curve of V. alginolyticus 178 in the presence of CLPs was tested according to previous methods with minor modifications (32). Briefly, a single colony of V. alginolyticus 178 was transferred into 5 ml of Zobell 2216E liquid medium and grown overnight.…”

Section: Methodsmentioning

confidence: 99%

Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

Xiu

Zhang

et al. 2017

“…It is highly probable that CPS/EPS production in the members of the MMC is tightly regulated, considering the importance of these products for bacterial survival in various ecological niches. In many bacteria, CPS/EPS regulation may depend on promoters (34) and external factors such as oxygen availability (35) or quorum sensing (36). Similar studies would be needed to decipher the regulatory mechanisms at stake in mycoplasmas.…”

Section: Discussionmentioning

confidence: 99%

Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Bertin

Pau-Roblot

Courtois

et al. 2015

iMycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a ␤(1¡6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of ␤(1¡2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides. Within the class Mollicutes, the so-called Mycoplasma mycoides cluster (MMC) (1) is unique: all the members of the MMC are pathogenic for ruminants, yet from a phylogenetic point of view, this cluster belongs to the Spiroplasma clade, which includes a number of species isolated from plants or insects (2). The taxonomy of the MMC was modified recently to reflect more precisely the phylogeny of this group. Because all the cluster members are closely related, their 16S rRNA gene sequences did not provide sufficient resolution to discriminate them accurately (3). A multilocus sequence typing approach was used to obtain a more precise phylogeny (4), which led to a simplification of the taxonomy (5) ( Table 1). The cluster currently comprises five species or subspecies: Mycoplasma mycoides subsp. mycoides, which was known formerly as the "small colony" (SC) biotype (1), M. mycoides subsp. capri, M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, and M. leachii. In addition, M. mycoides subsp. capri is subdivided in two serovars: the "large colony" serovar (M. mycoides subsp...

“…The static biofilm assay was carried out as described elsewhere (25,26). P. aeruginosa PAO1 cells were grown to an OD 600 of 3 in LB broth with vigorous shaking.…”

Section: Methodsmentioning

confidence: 99%

Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

Kim

Park

Lee

2015

Anthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation of Pseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product of P. aeruginosa, had an opposite effect on P. aeruginosa biofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment of P. aeruginosa at the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole on P. aeruginosa biofilm formation. Biofilms are a representative example of bacterial group behavior that provides cells with many biological advantages, such as high infectivity, antibiotic resistance, and strong survivability (1, 2). Currently, most persistent bacterial infections are believed to be associated with antibiotic-resistant biofilms of pathogenic bacteria (3, 4). Pseudomonas aeruginosa, a Gram-negative bacterium, is a ubiquitous and major opportunistic human pathogen. The colonization and biofilm formation of P. aeruginosa cause great losses in many industrial facilities and serious infections such as cystic fibrosis, microbial keratitis, and burn wound infections in humans (4-8). Therefore, control of P. aeruginosa biofilms is a very important issue in medicine, public health, and industry.Anthranilate and indole are both aromatic compounds produced from tryptophan metabolism. In bacteria, tryptophan is metabolized differently depending on the bacterial species, and the key enzyme of this differentiation is tryptophanase, encoded by the tnaA gene (9). Many bacteria, such as Escherichia coli, Haemophilus influenzae, and Vibrio vulnificus, produce indole from tryptophan, since they have tnaA, which converts tryptophan into indole, pyruvate, and ammonia (9, 10). How...