Modulation of Biofilm-Formation in <i>Salmonella enterica</i> Serovar Typhimurium by the Periplasmic DsbA/DsbB Oxidoreductase System Requires the GGDEF-EAL Domain Protein STM3615

<div><p>In <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3′, 5′-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either <i>dsbA</i> or <i>dsbB</i>, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a <i>r</i>ed, <i>d</i>ry <i>a</i>nd <i>r</i>ough (<i>rdar</i>) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting <i>csgD</i> in either <i>dsb</i> mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the <i>dsb</i> mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene <i>STM3615 (yhjK)</i>, previously not connected to <i>rdar</i> morphotype development, also abrogated the escalated <i>rdar</i> morphotype formation in <i>dsb</i> mutant backgrounds. Enhanced biofilm-formation in <i>dsb</i> mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both <i>dsb</i> mutants initially showed an escalated <i>rdar</i> morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in <i>S.</i> Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators.</p></div

Effect of <i>ydiV</i>, <i>yhjK</i> and <i>yhjH</i> mutations on <i>dsb</i> associated biofilm-formation.

<p>Immunoblot for CsgD from overnight salt-less Luria Agar cultures shows altered level of CsgD expression for <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants by introducing mutations in <i>ydiV</i>, <i>yhjK, ydiV-yhjK</i>, and <i>yhjH</i> genes respectively.</p

DsbA/B regulatory pathway leading to <i>rdar</i> morphotype formation in <i>S.</i> Typhimurium.

<p>In the illustration, the OM indicates outer cell membrane and IM refers inner cell membrane. The diguanyl cyclases are marked as black and the phosphodiesterases are marked as grey.</p

Biofilm-formation on solid media and liquid media.

<p>The formation of <i>rdar</i> morphotype in wild type, single and double <i>dsb</i> mutants grown for 48 hours on Congo red plates at 28°C is illustrated in panel (<b>A</b>). This escalated rdar morphotype development can be reverted to wild type level by <i>trans</i>-complementation with corresponding cloned <i>dsb</i> genes, or by introducing a cloned <i>yhjH</i> gene or depleting <i>csgD</i> (<b>B</b>). VC indicates the vector control, pBAD30. <b>C</b>) Crystal violet staining of biofilm adherent to polystyrene tubes as an indicator of biofilm-formation at liquid-air interface. The <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants fail to make a pellicle at air-liquid interface in static LB without salt culture at 28°C 24 hours post inoculation. <b>D</b>) Quantification of adherent biofilm measured as retained Crystal violet in biofilm. Error bars indicate SEM. ***  = p≤0.001.</p

Effect of <i>ΔdsbA</i> and <i>ΔdsbB</i> mutations on the expression of CsgA and CsgD.

<p><b>A</b>) Coomassie blue stained SDS-PAGE gel revealing increased CsgA production in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants when grown on LA without salt at 28°C at 24 hours of incubation. <b>B</b>) Immunoblot for CsgD shows increased level of CsgD for <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants from the same bacterial cultures. Numbers underneath the lanes in <b>A</b>) and <b>B</b>) specifies the relative band intensities. The <i>csgD-lacZ</i> promoter fusion activity at 18 hours post inoculation from salt-less Luria Agar cultures (<b>C</b>) or from salt-less Luria broth cultures (<b>D</b>) grown at 28°C. Error bars indicate SEM. ***  = p≤0.001; **  = p≤0.01; ns  =  not significant compared to respective wild type.</p

Strains and Plasmids.

<p>Strains and Plasmids.</p

Effect of reducing stress on biofilm.

<p><b>A</b>) Upon reductive stress (5 mM DTT) <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants first shows an escalated <i>rdar</i> morpotype development, where after the appearance becomes smoother and mucoid. Overexpression of YhjH and deletion of <i>csgD</i> enhances slime production in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants under DTT reductive stress. Coomassie blue stained SDS-PAGE (<b>B</b>) and immunoblot (<b>C</b>) show decrease in the amounts of respectively CsgA and CsgD under 5 mM DTT stress from 18 hours at 28°C. The numbers underneath the lanes indicate relative amounts. <b>D</b>) and <b>E</b>) The <i>csgD-lacZ</i> promoter fusion activity in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants under 5 mM DTT stress in LB and LA cultures at 28°C. Error bars indicate SEM. ns  =  non-significant compared to respective wild type.</p

Effect of GGDEF/EAL protein gene mutations on <i>dsb</i> associated biofilm-formation.

<p>The development of <i>rdar</i> morphotype in wild type, single and double mutants on Congo red plates at 28°C after 24 and 48 hours of incubation.</p

Effect of GGDEF/EAL proteins on <i>dsb</i> associated biofilm-formation.

<p>−/+/2+/3+ etc. represents the degree of the biofilm-formation compared to the WT.</p

Competition assay of the Δ<i>greA</i>Δ<i>greB</i> mutant strain versus WT.

<p>Amount of bacteria in liver and spleen was determined at 4 days post infection. A total of ~2E+7 colony forming units (cfu) of WT and the Δ<i>greA</i>Δ<i>greB</i> strain at a 1:1 ratio was administered orally to 5 mice.</p