CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

Abstract

Plague-causing Yersinia pestis is transmitted through regurgitation when it forms a biofilm-mediated blockage in the foregut of its flea vector. This biofilm is composed of an extracellular polysaccharide substance (EPS) produced when cyclic-di-GMP (c-di-GMP) levels are elevated. The Y. pestis diguanylate cyclase enzymes HmsD and HmsT synthesize c-di-GMP. HmsD is required for biofilm blockage formation but contributes minimally to in vitro biofilms. HmsT, however, is necessary for in vitro biofilms and contributes to intermediate rates of biofilm blockage. C-di-GMP synthesis is regulated at the transcriptional and posttranscriptional levels. In this, the global RNA chaperone, Hfq, posttranscriptionally represses hmsT mRNA translation. How c-di-GMP levels and biofilm blockage formation is modulated by nutritional stimuli encountered in the flea gut is unknown. Here, the RNA-binding regulator protein CsrA, which controls c-di-GMP-mediated biofilm formation and central carbon metabolism responses in many Gammaproteobacteria, was assessed for its role in Y. pestis biofilm formation. We determined that CsrA was required for markedly greater c-di-GMP and EPS levels when Y. pestis was cultivated on alternative sugars implicated in flea biofilm blockage metabolism. Our assays, composed of mobility shifts, quantification of mRNA translation, stability, and abundance, and epistasis analyses of a csrA hfq double mutant strain substantiated that CsrA represses hfq mRNA translation, thereby alleviating Hfq-dependent repression of hmsT mRNA translation. Additionally, a csrA mutant exhibited intermediately reduced biofilm blockage rates, resembling an hmsT mutant. Hence, we reveal CsrA-mediated control of c-di-GMP synthesis in Y. pestis as a tiered, posttranscriptional regulatory process that enhances biofilm blockage-mediated transmission from fleas. IMPORTANCE Yersinia pestis, the bacterial agent of bubonic plague, produces a c-di-GMP-dependent biofilm-mediated blockage of the flea vector foregut to facilitate its transmission by flea bite. However, the intricate molecular regulatory processes that underlie c-di-GMP-dependent biofilm formation and thus, biofilm-mediated blockage in response to the nutritional environment of the flea are largely undefined. This study provides a novel mechanistic understanding of how CsrA transduces alternative sugar metabolism cues to induce c-di-GMP-dependent biofilm formation required for efficient Y. pestis regurgitative transmission through biofilm-mediated flea foregut blockage. The Y. pestis-flea interaction represents a unique, biologically relevant, in vivo perspective on the role of CsrA in biofilm regulation.

Keywords: Xenopsylla cheopis fleas; Yersinia pestis; c-di-GMP; carbon storage regulator.

FIG 1

CsrA is required for EPS production and c-di-GMP synthesis. (A) Congo red (CR) binding assays were used to quantify EPS production of strains cultured in HIB or TMH supplemented with 0.2% of glucose (TMH-glu), galactose (TMH-gal), or ribose (TMH-rib). Error bars represent the means ± standard errors of the means (SEM) of bound CR samples from 3 to 6 independent experiments. (B) C-di-GMP was extracted from strains grown in TMH-glu, TMH-gal, or TMH-rib. Means of two independent experiments are shown. Statistical significance was determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons posttest for each medium type (black lines) or to compare the WT strain across media (gray lines) (*, P < 0.05; ***, P,  0.0005; ****, P < 0.0001; n.s., not significant).

FIG 2

CsrA does not directly regulate hmsT mRNA translation. (A) Schematic of the inducible GFP fusion reporters in which the native upstream untranslated sequences and the first 9 or 10 codons of gyrB (negative control), flhDC (positive control), hmsP, and hmsT were fused in frame to gfpmut3.1 (gfp) and the anhydrotetracyline (ATc)-inducible promoter P_tetO in the WT and ΔcsrA strains. (B) Posttranscriptional fusion reporter strains grown in TMH-gal to log phase were induced with ATc. At 3 h postinduction, the relative fluorescent units (RFU) and OD₆₀₀ were measured. Uninduced RFU/OD₆₀₀ values were subtracted from induced RFU/OD₆₀₀ values to compare between strains. Error bars represent means ± SEM from three independent experiments. Statistical significance was determined by an unpaired t test (*, P < 0.05; n.s., not significant). (C) Steady-state transcript levels of hmsP and hmsT were compared between the WT and ΔcsrA strains. Means ± SD from two independent experiments are shown. Statistical significance was determined with a Student's t test (*, P < 0.05; n.s., not significant). For gel mobility shift assays, 0.8 nM 3′biotin end-labeled hns (negative control) (D), flhDC (positive control) (E), or hmsT (F) probes were incubated with increasing concentrations of purified CsrA-His₆. Kerafast biotinylated sRNA ladder (L), free (Fr), and bound (Bo) species are indicated. A broken line indicates migration of Fr labeled probe. One representative of two independent experiments is shown.

FIG 3

CsrA negatively regulates hfq mRNA translation. (A) Nucleotide sequence of the 3′ end of the miaA gene (shaded gray) and the intercistronic region miaA-hfq genes plus ATG start of the hfq mRNA. The Shine-Dalgarno (SD) sequence and the ATG start codon for the hfq mRNA coding region are boldfaced and underlined. GGA motifs of the two putative CsrA binding sites are in red. The transcriptional start site (TSS) from the immediate upstream promoter is depicted by an arrow. (B) Posttranscriptional fusion reporter plasmids composing the upstream sequence and the first 9 codons of hfq fused to gfpmut3.1 (gfp), and the P_tetO promoter was used in the WT and ΔcsrA strains. Error bars represent mean ± SEM relative RFU/OD₆₀₀ from four independent experiments.

FIG 4

CsrA binds to two GGA binding sites in the 5′ UTR of hfq mRNA. (A) 3′Biotin end-labeled hfq probe (0.8 nM) and increasing concentrations of purified CsrA-His₆ were coincubated. Kerafast biotinylated sRNA ladder (L), free (Fr), and bound (Bo) species are indicated. A broken line indicates migration of Fr labeled probe. (B) For mobility shift competition assays, labeled hfq probe was coincubated with unlabeled hfq probe at 2 (1.6 nM) or 10 (8 nM) times more than the labeled hfq probe and 112.5 nM CsrA-His₆. (C, D, and E) Binding site mutant mobility shift assays were performed as described for panel A, except the hfq-labeled probes contained a mutation at BS1 (C), BS2 (D), or BS1/BS2 (E). One representative of two independent experiments is shown.

FIG 5

mRNA stability and translation of the hfq mRNA. (A) The WT and ΔcsrA strains were grown to log phase in TMH-gal, and RNA was isolated from samples taken at 0 min (prerifampin) and after the addition of 400 μg/ml rifampin. Relative hfq mRNA levels remaining at the indicated time points were quantified using RT-qPCR. The amount of hfq mRNA in each strain at 0 min relative to rifampin addition was set to 100%. The percent mRNA remaining thereafter was plotted versus time as semilogarithmic graphs. The mean ± SEM percent mRNA from four independent experiments is shown. (B) In vitro translational assays were performed with the PURExpress kit using translational fusions transcripts of hfq-gfp and hmsT-gfp (negative control) expressed from a T7 promoter. The mean ± SD fold change in HmsT-GFP and Hfq-GFP signal between samples in the presence or absence of CsrA was derived from three technical replicates of the immunodot blot. One representative dot blot is shown.

FIG 6

Epistasis analysis confirms that CsrA indirectly derepresses hmsT mRNA translation by targeting the hfq mRNA. (A) hmsT mRNA stability was determined as described for Fig. 5A. (B) Steady-state transcript levels of hmsT were compared between the WT (same as Fig. 2C), ΔcsrA (same as Fig. 2C), ΔcsrA Δhfq, and complemented ΔcsrA Δhfq (pLGhfq) strains. Means ± SD from two independent experiments are shown. (C) Congo red (CR) binding assays were used to quantify EPS production in strains cultured in LB medium. A representative picture of strains grown for 48 h on LB containing CR is shown below. Error bars represent the mean ± SD bound CR from two independent experiments. Statistical significance was determined using one-way ANOVA with a Dunnett’s multiple-comparison posttest (**, P < 0.01; ***, P = 0.0005; n.s., not significant).

FIG 7

Flea foregut blockage and infection dynamics of the ΔcsrA strain. Cohorts of Xenopsylla cheopis fleas were artificially infected with the WT (black), ΔcsrA (red), or ΔcsrA::csrA (gray) strains and monitored for blockage over 28 days postinfection (dpi). (A) Cumulative flea blockage rate of each strain is shown for 100 fleas (50 male, 50 female), with error bars representing the means ± SEM from three independent experiments. One-way ANOVA with Holm Sidak’s multiple-comparison posttest was used to determine statistical significance (*, P < 0.05; n.s., not significant). (B) Number of fleas blocked on 5, 8, 12, 15, 19, 22, and 26 dpi for each strain is shown as means ± SEM for three independent experiments. Two-way ANOVA with Holm Sidak’s multiple-comparison posttest was used to determine statistical significance for each time point (**, P < 0.01; ****, P < 0.0001). (C) Mean number of CFU per flea (n = 10 to 20) at 0, 7, and 28 dpi. Error bars represent means ± SEM from 2 to 3 independent experiments. Two-way ANOVA was used to test for statistical significance (n.s., not significant). (D) Percentage of fleas infected (n = 10 to 20) at 0, 7, and 28 dpi. Error bars represent means ± SEM from 2 to 3 independent experiments. Two-way ANOVA was used to test for statistical significance (n.s., not significant).

FIG 8

Current model for the CsrA-dependent regulation of c-di-GMP synthesis and biofilm formation in Y. pestis. In Y. pestis, the HmsHFRS proteins synthesize and export EPS to outside the cell to propagate biofilm formation, which leads to biofilm-mediated blockage in fleas. CsrA transduces alternative carbon metabolism cues to enhance biofilm EPS production (green thick broken arrow). CsrA does this through indirect positive regulation of the c-di-GMP synthesis enzyme, HmsT. Hfq represses hmsT mRNA translation by promoting its mRNA decay, but CsrA binds to two binding sites in the 5′UTR of hfq to inhibit its translation (solid red line), thereby alleviating repression of the hmsT mRNA translation by Hfq. How and under which physiological conditions ncRNAs CsrB and CsrC antagonize CsrA activity (solid blue line) in Y. pestis are yet to be determined.