Enteric helminths promote Salmonella co-infection by altering the intestinal metabolome

Intestinal helminth infections occur pre dominantly in regions where exposure to enteric bacterial pathogens is also common. Helminth infections inhibit host immunity against microbial pathogens, which has largely been attributed to the induction of regulatory or type 2 (Th2) immune responses. Here we demonstrate an additional three-way interaction in which helminth infection alters the metabolic environment of the host intestine to enhance bacterial pathogenicity. We show that an ongoing helminth infection increased colonization by Salmonella independently of T regulatory or Th2 cells. Instead, helminth infection altered the metabolic profile of the intestine, which directly enhanced bacterial expression of Salmonella pathogenicity island 1 (SPI-1) genes and increased intracellular invasion. These data reveal a novel mechanism by which a helminth-modified metabolome promotes susceptibility to bacterial co-infection

Manual Annotation, Transcriptional Analysis, and Protein Expression Studies Reveal Novel Genes in the agl Cluster Responsible for N Glycosylation in the Halophilic Archaeon Haloferax volcanii▿ †

While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described

Expression of AglQ mutants.

<p>The levels of the different versions of AglQ fused to GFP generated following site-directed mutagenesis and expression in Δ<i>aglQ</i> cells is shown. The protein content of equivalent amounts of <i>Hfx</i>. <i>volcanii</i> Δ<i>aglQ</i> cells expressing the various AglQ mutants were separated by SDS-PAGE subjected to immunoblot using anti-GFP and appropriate secondary HRP-conjugated antibodies. The positions of 55 and 40 kDa molecular weight markers are depicted on the right of each panel.</p

AglQ is a soluble protein.

<p><i>Hfx</i>. <i>volcanii</i> cells transformed to express GFP-AglQ were separated into membrane and cytosolic (supernatant) fractions and probed with anti-GFP (α-GFP) or anti-SRP54 (α-SRP54) antibodies, as was a total protein extract (cell). Alternatively, the position of the S-layer glycoprotein in the same fractions was identified by Coomassie staining. The proteins migrate with the following molecular masses in SDS-PAGE: S-layer glycoprotein, 180 kDa [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081782#B20" target="_blank">20</a>], SRP-54, 51 kDa [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081782#B16" target="_blank">16</a>], GFP-AglQ, 44 kDa.</p

DolP glycosylation in compromised in Δ<i>aglQ</i> cells.

<p>Normal phase LC-ESI MS analysis of a total <i>Hfx</i>. <i>volcanii</i> lipid extract revealed [M-H]^- ions corresponding to (A) C₅₅ and C₆₀ DolP, as well as the same lipids modified by (B) hexose (C₅₅ and C₆₀ DolP-hex). [M-2H]^2- ions corresponding to the same lipids modified by (C) hexose and a hexuronic acid (C₅₅ and C₆₀ DolP-hex-hexUA) and (D) hexose and two hexuronic acids (C₅₅ and C₆₀ DolP-hex-hexUA-hexUA) were also detected, as indicated. E. Δ<i>aglQ</i> cells do not contain tetrasaccharide-charged DolP, unlike cells of the parent strain, where [M-2H]^2- ions corresponding to DolP modified by hexose, two hexuronic acids and a methyl ester of hexuronic acid are seen (C₅₅ and C₆₀ DolP-tetrasaccharide) are readily detected (inset).</p

AglQ Is a Novel Component of the <i>Haloferax volcanii</i> N-Glycosylation Pathway

<div><p>N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile <i>Haloferax volcanii</i> have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of <i>Hfx. volcanii</i> glycoproteins, such as the S-layer glycoprotein. In cells deleted of <i>aglQ</i>, both dolichol phosphate, the lipid carrier used in <i>Hfx. volcanii</i> N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in <i>Hfx. volcanii</i> N-glycosylation. </p> </div

S-layer integrity in compromised in Δ<i>aglQ</i> cells.

<p>Parent strain (top panel) and Δ<i>aglQ</i> cells (lower panel) were challenged with 1 mg/ml proteinase K at 42°C. Aliquots were removed immediately prior to incubation with proteinase K and at 15-30 min intervals following addition of the protease for up to 3 h and examined by 7.5% SDS-PAGE and Coomassie staining. The S-layer glycoprotein band from each gel is presented.</p

Multiple Salmonella-pathogenicity island 2 effectors are required to facilitate bacterial establishment of its intracellular niche and virulence.

The pathogenesis of Salmonella Typhimurium depends on the bacterium's ability to survive and replicate within host cells. The formation and maintenance of a unique membrane-bound compartment, termed the Salmonella-containing vacuole (SCV), is essential for S. Typhimurium pathogenesis. SCV-bound S. Typhimurium induces formation of filamentous tubules that radiate outwards from the SCV, termed Salmonella-induced filaments (SIFs). SIF formation is concomitant with the onset of replication within host epithelial cells. SIF biogenesis, formation and maintenance of the SCV, and the intracellular positioning of the SCV within the host cell requires translocation of bacterial proteins (effectors) into the host cell. Effectors secreted by the type III secretion system encoded on Salmonella pathogenicity island 2 (T3SS2) function to interfere with host cellular processes and promote both intracellular survival and replication of S. Typhimurium. Seven T3SS2-secreted effectors, SifA, SopD2, PipB2, SteA, SseJ, SseF, and SseG have previously been implicated to play complementary, redundant, and/or antagonistic roles with respect to SIF biogenesis, intracellular positioning of the SCV, and SCV membrane dynamics modulation during infection. We undertook a systematic study to delineate the contribution of each effector to these processes by (i) deleting all seven of these effectors in a single S. Typhimurium strain; and (ii) deleting combinations of multiple effectors based on putative effector function. Using this deletion mutant library, we show that each of SIF biogenesis, intracellular SCV localization, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. Together, our data demonstrates the complex interplay between these seven effectors and highlights the necessity to study T3SS2-secreted effectors as groups, rather than studies of individual effectors

S-layer glycoprotein N-glycosylation in compromised in Δ<i>aglQ</i> cells.

<p>Following trypsin treatment, the S-layer glycoprotein of Δ<i>aglQ</i> cells was examined by normal phase LC-ESI MS. Shown are profiles obtained for the ¹ERGNLDADSESFNK¹⁴ glycopeptide. Arrows indicate the positions of [M+2H]²⁺ ions corresponding to the peptide modified by (A) the first, (B) the first two and (C) the first three sugar residues of the pentasaccharide normally N-linked to this position. No [M+2H]²⁺ ion peaks corresponding to the same peptide modified by the first four pentasaccharide residues (D) nor the complete pentasaccharide (E) were detected in the <i>aglQ</i> deletion strain, despite such species being readily detected in the same sample obtained from parent strain cells (insets of D and E, respectively). The identity of each pentasaccharide subunit is provided in the inset in (A).</p