Maintaining Integrity Under Stress:Envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria

The Gram-negative bacterial envelope is an essential interface between the intracellular and harsh extracellular environment. Envelope stress responses (ESRs) are crucial to the maintenance of this barrier and function to detect and respond to perturbations in the envelope, caused by environmental stresses. Pathogenic bacteria are exposed to an array of challenging and stressful conditions during their lifecycle and, in particular, during infection of a host. As such, maintenance of envelope homeostasis is essential to their ability to successfully cause infection. This review will discuss our current understanding of the σE- and Cpx-regulated ESRs, with a specific focus on their role in the virulence of a number of model pathogens

Examination of bacterial growth, cell shape maintenance, DNA segregation, and death in Caulobacter crescentus

Despite their long-standing role as comparatively simple model organisms, bacteria undergo very complex physiological processes. Their growth depends on the coordinated action of the peptidoglycan remodeling complexes, which include biosynthetic transpeptidases and transglycosylases as well as hydrolytic enzymes. While the roles of transpeptidases in specific modes of bacterial growth are well understood, this dissertation addresses the contribution of transglycosylases to the peptidoglycan synthesis in Caulobacter. We find that tranglycosylases are largely redundant, but the loss of CC0252 leads to a general defect in cell wall integrity. The remodeling complexes themselves are spatially controlled by the three-dimensional arrangements of cytoskeletal structures within the cells. In particular, elongation of most rod-shaped bacteria requires the actin homolog MreB, but despite the central importance of MreB to cell shape maintenance and viability, its regulation is poorly understood. We report the identification of MbiA, a small protein that genetically and biochemically interacts with MreB and whose overexpression mimics the MreB loss-of-function phenotype while also perturbing MreB localization. Bacterial chromosome segregation is also a complex, multistep process. We show that it progresses through several phases involving polar release of the origin, its slow initial motion, and the fast late motion dependent on the action of the polymer-forming ATPase ParA. Finally, we find that upon DNA damage, Caulobacter induces a BapE endonuclease and activates an apoptotic-like response, demonstrating the ability to undergo a process long thought to be the sole purview of eukaryotes

NlpD and AmiC localize to division sites independently of YraP and/or the Tol-Pal system.

<p>(<b>A-D</b>) Overnight cultures of (<b>A</b>) MT47 (^<i>ΔSS</i><i>nlpD</i>), (<b>B</b>) MT141 (^<i>ΔSS</i><i>nlpD ΔyraP</i>), (<b>C</b>) MT53 (^<i>ΔSS</i><i>nlpD ΔtolQ-pal</i>), or (<b>D</b>) MT147 (^<i>ΔSS</i><i>nlpD ΔtolQ-pal ΔyraP</i>) harboring the integrated expression construct attHKNP20 (<i>P</i>_<i>lac</i>::<i>nlpD-mCherry</i>) were diluted in minimal M9-maltose medium supplemented with 100μM IPTG. (<b>E-H</b>) Overnight cultures of (<b>E</b>) TB143 (<i>ΔamiC</i>), (<b>F</b>) MT150 (<i>ΔamiC ΔyraP</i>), (<b>G</b>) MT149 (<i>ΔamiC ΔtolQ-pal</i>), or (<b>H</b>) MT178 (<i>ΔamiC ΔtolQ-pal ΔyraP</i>) harboring the integrated expression construct attHKNP16 (<i>P</i>_{<i>lac-m3</i>}::<i>amiC-gfp</i>) were diluted in minimal M9-maltose medium supplemented with 25μM IPTG. For all strains, cells were grown at 30°C to an OD₆₀₀ of 0.2–0.3 before they were visualized on 2% agarose pads by phase contrast and fluorescence microscopy. In all images, arrows indicate the localization of the protein fusion to division sites. Bar = 4μm.</p

Structure-function analysis of NlpD.

<p>The domain organization of NlpD is illustrated. Indicated are the signal sequence (SS; yellow), lysin motif (LysM; blue), and the degenerate LytM domain (dLytM; green). Also shown are the NlpD truncations that were expressed under the control of the IPTG-inducible lactose promoter either as an untagged protein or as a C-terminal mCherry fusion. Truncations lacking ^SSNlpD are expressed as soluble periplasmic proteins fused to the DsbA signal peptide. Columns indicate (i) the NlpD residues present in each truncation, (ii) whether the fusion to mCherry accumulated at division sites strongly (+++), poorly (+), or appeared evenly distributed along the periphery of the cell (-), and (iii) whether the untagged truncation could (+) or could not (-) compensate for the loss of endogenous NlpD for proper cell separation. ND, not determined.</p

Recruitment of YraP to division sites is dependent on its OM localization but independent of NlpD, the Tol-Pal complex, and AmiC.

<p>(<b>A-C</b>) Overnight cultures of MT140 (<i>ΔyraP</i>) harboring the integrated construct (<b>A</b>) attλMT197 (<i>P</i>_<i>lac</i>::<i>yraP</i>^<i>WT</i><i>-mCherry</i>), (<b>B</b>) attλMT199 (<i>P</i>_<i>lac</i>::<i>yraP</i>^<i>IM</i><i>-mCherry</i>), or (<b>C</b>) attλMT210 (<i>P</i>_<i>lac</i>::^<i>ss</i><i>dsbA-yraP</i>^<i>peri</i><i>-mCherry</i>) were diluted in minimal M9-maltose medium supplemented with 10μM (<b>A-C</b>) or 20μM (<b>B</b>) IPTG. (<b>D-G</b>) Overnight cultures of (<b>D</b>) MT140 (<i>ΔyraP</i>), (<b>E</b>) MT141 (<i>ΔyraP</i> ^<i>ΔSS</i><i>nlpD</i>), (<b>F</b>) MT147 (<i>ΔyraP</i> ^<i>ΔSS</i><i>nlpD ΔtolQ-pal</i>), and (<b>G</b>) AAY22 (<i>ΔyraP ΔamiC</i>) harboring the integrated construct attλMT197 (<i>P</i>_<i>lac</i>::<i>yraP</i>^<i>WT</i><i>-mCherry</i>) were diluted in minimal M9-maltose medium supplemented with 10μM IPTG. All cultures were grown at 30°C to an OD₆₀₀ of 0.15–0.2 before cells were visualized on 2% agarose pads by phase contrast and fluorescence microscopy. Arrows indicate localization of the protein fusion to division sites. Bar = 4μm.</p

OM localization of NlpD is required for proper cell separation.

<p>(<b>A</b>) The domain structure of NlpD is illustrated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006888#pgen.1006888.g001" target="_blank">Fig 1</a>. Details of the signal sequence are presented with the lipobox in red and the arrow indicating the cleavage site just before the acylated cysteine. The IM-retained variant (NlpD^(S27D)) contains a mutated signal sequence (indicated by the asterisk) with an aspartate at the +2 position after the acylated cysteine (underlined). The soluble periplasmic variant (NlpD^(27–379)) is fused to the DsbA signal peptide (purple) that is cleaved upon export to the periplasm via the Sec system. (<b>B-D</b>) Cytological assay to determine the subcellular localization of the NlpD variants. Overnight cultures of MT47 (^<i>ΔSS</i><i>nlpD</i>) expressing different NlpD-mCherry fusions from the integrated constructs (<b>B</b>) attHKNP20 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^<i>WT</i><i>-mCherry</i>), (<b>C</b>) attHKMT21 (<i>P</i>_<i>lac</i>::<i>nlpD</i> ^{<i>(S27D)</i>}<i>-mCherry</i>), or (<b>D</b>) attHKMT147 (<i>P</i>_<i>lac</i>::^<i>ss</i><i>dsbA-nlpD</i> ^{<i>(27–379)</i>}<i>-mCherry</i>) were diluted in minimal M9-maltose medium supplemented with 25 μM (<b>D</b>) or 150 μM (<b>B, C</b>) IPTG. Cells were grown at 30°C to an OD₆₀₀ of 0.4, washed and then osmotically shocked by resuspension in plasmolysis buffer and the plasmolyzed cells were visualized by phase contrast and fluorescence microscopy. Arrows indicate signals that display a smooth OM peripheral signal in (<b>B</b>), track with the inner membrane in (<b>C</b>), or fill the increased periplasmic spaces of plasmolysis bays in (<b>D</b>). Bar = 4μm. (<b>E-G</b>) Overnight cultures of MT50 (^<i>ΔSS</i><i>nlpD ΔenvC</i>) harboring the integrated expression constructs (<b>E</b>) attHKMT20 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^<i>WT</i>), (<b>F</b>) attHKMT12 (<i>P</i>_<i>lac</i>::<i>nlpD</i> ^{<i>(S27D)</i>}), or (<b>G</b>) attHKMT121 (<i>P</i>_<i>lac</i>::^<i>ss</i><i>dsbA-nlpD</i> ^{<i>(27–379)</i>}) were diluted in minimal M9-maltose medium and grown at 37°C until mid-log. Cultures were then backdiluted into M9-maltose medium without (grey histogram) or with 150 μM (blue histogram) or 1 mM (red histogram) IPTG and grown at 37°C to an OD₆₀₀ of 0.2–0.3 before flow cytometry analysis. (<b>H</b>) Cells of MT122 (^<i>ΔSS</i><i>nlpD</i>) and MT123 (^<i>ΔSS</i><i>nlpD ΔamiC</i>) alone or harboring the integrated constructs described above were grown and spotted on CPRG agar as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006888#pgen.1006888.g003" target="_blank">Fig 3I</a>. The subcellular localization of each NlpD variant is indicated in the square brackets: OM, outer membrane; IM, inner membrane; peri, periplasm.</p

Localization of YraP or NlpD in cephalexin-treated cells.

<p>Overnight cultures of (<b>A-B</b>) MT140 (<i>ΔyraP</i>) harboring the integrated construct attλMT197 (<i>P</i>_<i>lac</i>::<i>yraP-mCherry</i>) or (<b>C-D</b>) MT47 (^<i>ΔSS</i><i>nlpD</i>) harboring the integrated expression construct attHKNP20 (<i>P</i>_<i>lac</i>::<i>nlpD-mCherry</i>) were diluted in minimal M9-maltose medium supplemented with either 25μM (<b>A-B</b>) or 100μM (<b>C-D</b>) IPTG and grown at 30°C until mid-log. Cultures were then backdiluted into M9-maltose medium with the indicated IPTG concentration with or without 10μg/ml cephalexin as indicated. Cells were grown at 30°C to an OD₆₀₀ of 0.2 before they were visualized on 2% agarose pads by phase contrast and fluorescence microscopy. Arrows indicate localization of the protein fusion to division sites. Bar = 10μm.</p

Only full-length NlpD properly promotes cell separation.

<p>(<b>A-C</b>) MT50 (^<i>ΔSS</i><i>nlpD ΔenvC</i>) or TB140 (<i>ΔenvC</i>) cells were grown in minimal M9-maltose medium at 37°C to an OD₆₀₀ of 0.2–0.3 before visualization on 2% agarose pads with DIC optics (<b>A-B</b>) or analyzed by flow cytometry (<b>C</b>). Bar = 10μm. (<b>D-H</b>) Overnight cultures of MT50 harboring the integrated expression constructs (<b>D</b>) attHKMT20 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^<i>WT</i>), (<b>E</b>) attHKMT102 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^{<i>(1–189)</i>}), (<b>F</b>) attHKMT104 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^{<i>(1–115)</i>}), (<b>G</b>) attHKMT179 (<i>Pl</i>_<i>ac</i>::^<i>ss</i><i>dsbA-nlpD</i>^{<i>(250–379)</i>}), or (<b>H</b>) attHKMT181 (<i>P</i>_<i>lac</i>::^<i>ss</i><i>dsbA-nlpD</i>^{<i>(189–379)</i>}) were diluted in minimal M9-maltose medium and grown at 37°C. Mid-log cultures were then backdiluted into M9-maltose medium with or without the inducer IPTG. Cells were further grown at 37°C to an OD₆₀₀ of 0.2–0.3 before flow cytometry analysis. Histograms of cultures that were either uninduced (grey) or induced with 150μM (<b>D, G-H</b>, blue) or 1mM (<b>E-F</b>, red) IPTG were overlayed. (<b>I</b>) Cells of MT122 (^<i>ΔSS</i><i>nlpD</i>) and MT123 (^<i>ΔSS</i><i>nlpD ΔamiC</i>) alone or harboring the integrated constructs attHKMT20 (<i>P</i>_<i>lac</i>::<i>nlpD</i>^<i>WT</i>), attHKMT179 (<i>Pl</i>_<i>ac</i>::^<i>ss</i><i>dsbA-nlpD</i>^{<i>(250–379)</i>}), or attHKMT181 (<i>P</i>_<i>lac</i>::^<i>ss</i><i>dsbA-nlpD</i>^{<i>(189–379)</i>}) were grown in LB at 30°C. Following normalization for cell density (OD₆₀₀ = 0.5), 5 μl of the resulting cultures was spotted on LB agar containing 150μM IPTG and 20 μg/ml CPRG. The plates were incubated at 30°C and photographed after 14 hours.</p

Model for the regulation of NlpD during cell division.

<p>Shown is a schematic depicting two cycles of a potential coupling mechanism coordinating septal PG splitting by NlpD/AmiC with OM invagination. Prior to the initiation of constriction, we envision that the dLytM domain of OM-anchored NlpD is prevented from accessing the PG layer and/or AmiC, potentially by physical distance or protein conformation constraints. When the divisome is activated, OM constriction promoted by the Tol-Pal system may bring the dLytM domain of NlpD into proximity of the PG layer and AmiC where it can stimulate septal PG splitting. AmiC activation also appears to require YraP, which may activate NlpD directly or indirectly through its yet to be determined role in maintaining OM integrity. PG processing by AmiC is expected to increase the distance between the OM and PG layer, thus returning NlpD to its inactive configuration and requiring another round of OM constriction to trigger further PG processing and so on until cell division is complete and the daughter cells are separated. Shown are hypothetical protein conformations and interactions within the Tol-Pal system based on current literature [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006888#pgen.1006888.ref065" target="_blank">65</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006888#pgen.1006888.ref071" target="_blank">71</a>].</p