The broadly conserved regulator PhoP links pathogen virulence and membrane potential in Escherichia coli

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87159/1/j.1365-2958.2011.07804.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/87159/2/MMI_7804_sm_FigS1-4-TabS1.pd

Multicellular Bacteria Deploy the Type VI Secretion System to Preemptively Strike Neighboring Cells

<div><p>The Type VI Secretion System (T6SS) functions in bacteria as a contractile nanomachine that punctures and delivers lethal effectors to a target cell. Virtually nothing is known about the lifestyle or physiology that dictates when bacteria normally produce their T6SS, which prevents a clear understanding of how bacteria benefit from its action in their natural habitat. <i>Proteus mirabilis</i> undergoes a characteristic developmental process to coordinate a multicellular swarming behavior and will discriminate itself from another <i>Proteus</i> isolate during swarming, resulting in a visible boundary termed a Dienes line. Using transposon mutagenesis, we discovered that this recognition phenomenon requires the lethal action of the T6SS. All mutants identified in the genetic screen had insertions within a single 33.5-kb region that encodes a T6SS and cognate Hcp-VrgG-linked effectors. The identified T6SS and primary effector operons were characterized by killing assays, by construction of additional mutants, by complementation, and by examining the activity of the type VI secretion system in real-time using live-cell microscopy on opposing swarms. We show that lethal T6SS-dependent activity occurs when a dominant strain infiltrates deeply beyond the boundary of the two swarms. Using this multicellular model, we found that social recognition in bacteria, underlying killing, and immunity to killing all require cell-cell contact, can be assigned to specific genes, and are dependent on the T6SS. The ability to survive a lethal T6SS attack equates to “recognition”. In contrast to the current model of T6SS being an offensive or defensive weapon our findings support a preemptive mechanism by which an entire population indiscriminately uses the T6SS for contact-dependent delivery of effectors during its cooperative mode of growth.</p></div

Infiltration of resistant and sensitive opposing swarms by <i>P. mirabilis</i> HI4320 expressing VipA::sfGFP.

<p>(<b>A</b>) Agar plate inoculated with <i>P. mirabilis</i> HI4320 VipA::sfGFP opposing strains HI4320 or mutant 9C1 expressing dsRED. Dienes lines (white arrows) formed between HI4320 VipA::sfGFP or HI4320 dsRED and 9C1 dsRED but not between HI4320 VipA::sfGFP and HI4320 dsRED. The fields examined by fluorescence microscopy (white boxes) are indicated at the intersection of the swarms. Active infiltration of (<b>B</b>) HI4320 dsRED and (<b>C</b>) mutant 9C1 dsRED by HI4320 expressing VipA::sfGFP. The numbered panels in (B) and (C) correspond to the numbered areas indicated in (A) and were viewed directly on the agar plate at the time of intersection. For each field, the individual green and red channels from the merged image are shown to maximize visualization of the infiltrating swarm. In (B) and (C) the panels boxed with a yellow border show dsRED-expressing bacteria infiltrating into the HI4320 VipA::sfGFP swarm are only observable with HI4320 dsRED (B1 and B2 red); the susceptible 9C1 dsRED is undetectable (C6 and C7 red). (<b>D</b>) Infiltrating HI4320 expressing VipA::sfGFP demonstrate numerous areas of T6SS activity (green) when in direct contact with target 9C1 cells expressing dsRED. Elapsed time (T) is indicated in seconds. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s012" target="_blank">Movie S3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s013" target="_blank">S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s014" target="_blank">S5</a>.</p

Contact-dependent preemptive antagonism is dependent on the T6SS.

<p>(<b>A</b>) A Dienes line (black arrows) forms between two different wild-type isolates, HI4320 and BB2000 (strain A and B kill each other). Loss of the T6SS (ΔT6) in either isolate by disruption of PMI0742 does not affect the discriminatory Dienes line (strain A kills strain B or strain B kills strain A). Loss of the T6SS in both isolates allows non-identical swarms to merge and the lack of T6SS-dependent killing appears as recognition (white arrow). (<b>B</b>) Mutant 9C1 maintains the ability to form a Dienes line with non-identical BB2000 lacking a T6SS (dashed arrow). No line appears when HI4320 and BB2000 both lack the T6SS (white arrow). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s005" target="_blank">Figure S5</a>.</p

The T6SS and primary effector operon are required for killing.

<p>The T6SS and primary effector operon are required for killing.</p

Visualization of the T6SS activity during multicellular infiltration and killing.

<p>(<b>A</b>) Actively swarming <i>P. mirabilis</i> HI4230 cells expressing VipA::sfGFP viewed under phase contrast and by fluorescence microscopy. Punctate green staining represents sites of T6SS assembly (arrows). (<b>B</b>) HI4320 and mutant 9C1 are observed merging at their swarm fronts on an agar surface (arrows) under phase contrast. (<b>C</b>) HI4320 VipA::sfGFP is observed infiltrating deep into the opposing 9C1 swarm expressing dsRED. Short arrows indicate individual HI4320 swarm cells within the 9C1 swarm. (<b>D</b>) Merged image of (B) and (C). The boxed region encapsulates intense green straining representing increased sfGFP-signal due to increased assembly of the T6SS sheath (VipA::sfGFP) by the front swarm edge of wild-type HI4320. (<b>E</b>) Visualization of individual HI4320 wild-type swarmer cells (green) that have infiltrated and are demonstrating multicellular swarming within the susceptible 9C1 swarm (red). (<b>F</b>) The forward movement of one individual HI4320 swarm cell over 30 seconds is indicated by yellow asterisk. Elapsed time (T) in seconds is indicated. Each frame in (E, F) represents 3 seconds elapsed time and the white bar is 50 µM. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s001" target="_blank">Figure S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s002" target="_blank">S2</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s010" target="_blank">Movie S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s011" target="_blank">S2</a>.</p

The primary <i>hcp-vgrG</i> T6SS effector operon encodes both killing and immunity functions.

<p>(<b>A</b>) Transcription of the primary effector operon based upon RT-PCR. Intergenic primer pairs (thin black arrows) flanking the open reading frames of PMI0750-PMI0759 are represented by letters. For each reaction (+) cDNA made from RNA using reverse transcriptase, (−) no RT enzyme, or (g) genomic DNA purified from HI4320 were used as template for PCR. (<b>B</b>) Predicted structural homology and potential functions for the effectors and other proteins encoded within the primary <i>hcp-vgrG</i> operon. Functional domains are color coded beneath each gene. (<b>C</b>) Wild-type HI4320, transposon mutants 9C1 and 12B5, and mutant <i>0758::kan</i> expressing pBAD empty vector or pBAD containing the indicated genes were inoculated onto agar containing 10 mM L-arabinose opposing mutant 9C1 containing empty vector. The presence of a Dienes line (+) indicates killing of 9C1. Mutant 9C1 expressing the same constructs were also examined against wild-type HI4320 for complementation (+) of the 9C1 immunity defect as indicated by absence of a Dienes line (immunity to HI4320). (<b>D–G</b>) <i>P. mirabilis</i> HI4320 and mutant 9C1 (center) were assessed for Dienes line formation against 9C1 with pBAD containing the indicated genes. PMI0756 is necessary and sufficient to restore 9C1 immunity against parental HI4320 (white arrows), while all three genes encoded by PMI0756, PMI0757, and PMI0758 are necessary and sufficient to restore both 9C1 immunity against HI4320 and 9C1 killing of 9C1 lacking PMI0756 (black arrows). (<b>H, I</b>) Mutant 12B5 (peripheral swarms) contains a transposon insertion in PMI0757 and is unable to kill mutant 9C1 unless 12B5 is complemented with pBAD containing PMI0757 and PMI0758. Parental HI4320 does not form a Dienes line with mutant 12B5 (pBAD). Complementation of 12B5 and the resulting Dienes line formation with 9C1 are indicated with black arrows in (H) and (I). All plates in (D–I) contain 10 mM L-arabinose and swarms labeled HI4320, 9C1, and 12B5 contain pBAD empty vector. (<b>J</b>) HI4320 lacking PMI0758 (0758::<i>kan</i>) carrying an uninduced clone of PMI0758 on pBAD does not form a Dienes line with mutant 9C1 in the absence of arabinose (−). (<b>K</b>) The ability to form a Dienes line with mutant 9C1 is fully restored by arabinose induction of the pBAD promoter (+) to express PMI0758 (<i>0758::kan</i> +PMI0758). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s003" target="_blank">Figure S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003608#ppat.1003608.s004" target="_blank">S4</a>.</p