J Bacteriol Virol.  2015 Mar;45(1):26-35. 10.4167/jbv.2015.45.1.26.

Cyclic AMP and Cyclic AMP-Receptor Protein are Required for Optimal Capsular Polysaccharide Expression

• Cho HK
• Shin SH

• KMID: 2168701
• DOI: http://doi.org/10.4167/jbv.2015.45.1.26

Abstract

Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.

Keyword

Vibrio vulnificus; Glucose; Adenylate cyclase; Cyclic AMP-receptor protein; Capsular polysaccharide

MeSH Terms

Adenylyl Cyclases
Complement System Proteins
Cyclic AMP Receptor Protein
Cyclic AMP*
Glucose
Metabolism
Operon
Vibrio vulnificus
Complement System Proteins
Cyclic AMP
Cyclic AMP Receptor Protein
Glucose

Figure

• 

  Figure 1. Colony morphologies of Vibrio vulnificus strains under different conditions. V. vulnificus strains were spread on the surfaces of HI agars or HI agars containing 100 μM cAMP, and cultured for 24 h at 37°C. Wild-type: M06-24/O, Wza-: RC398 containing mutated wza, Cya-: RC386 containing mutated cya, Cya+: RC390 containing complemented cya, Crp-: CMM710 containing mutated crp, Crp+: CMM714 containing complemented crp.

• 

  Figure 2. Effect of cyaA or crp mutation on chlorine resistance. For each V. vulnificus strain, approximately 105 mid-exponentialphase cells were exposed to either phosphate-buffered saline (PBS, pH 7.2) or 1.5 μg/ml NaOCl for 5 min, which was followed by dilution and plating. The experiments were conducted at least three times, and viable cell count was expressed as the log mean (large bar) and standard deviation (error bar) of triplicate measurements (*: p < 0.05 in two-way ANOVA). Wild-type: M06-24/O, Crp-: CMM710 containing mutated crp, Crp+: CMM714 containing complemented crp, Cya-: RC386 containing mutated cya, Cya+: RC390 containing complemented cya.

• 

  Figure 3. Effect of Crp on the expression of group 1 CPS operon. The three strains containing the Pwza::lacZ fusion construct, that is, RC342 containing wild-type crp (Wild-type), RC344 containing mutated crp (Crp-) and RC360 containing complemented crp (Crp-), were cultured in HI broths for 24 h at 37°C. Bacterial growths were determined by measuring optical densities of culture aliquots at 600 nm (OD600), and wza transcription was quantified by measuring β-galactosidase activity on a per cell basis (Miller units) in culture aliquots. Bacterial growth levels and β-galactosidase activities are the means and standard deviations (error bars) of triplicate measurements (*: p < 0.05 in Student's t-test).

• 

  Figure 4. Effect of glucose on the colony opacity of Vibrio vulnificus in the absence of Crp. CMM714 containing mutated crp was spread on the surface of TES-HI agars containing 0.0 (a) or 0.25% (b) glucose and cultured at 37°C overnight.

• 

  Figure 5. Effect of glucose on Vibrio vulnificus growth (A) and the expression of group 1 CPS operon (B) in the absence of Crp. RC344 containing mutated crp and the Pwza::lacZ fusion construct was cultured in TES-HI broths containing 0.0 or 0.25% glucose for 24 h at 37°C. Growth and wza transcription were determined and expressed as described in the legend of Fig. 2 (*: p < 0.05 in Student's t-test).

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