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.

Enteric Helminths Promote Salmonella Coinfection by Altering the Intestinal Metabolome.

Intestinal helminth infections occur predominantly 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 3-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 coinfection.

Nutrient Deprivation Affects Salmonella Invasion and Its Interaction with the Gastrointestinal Microbiota.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne enteric pathogen and a major cause of gastroenteritis in humans. It is known that molecules derived from the human fecal microbiota downregulate S. Typhimurium virulence gene expression and induce a starvation-like response. In this study, S. Typhimurium was cultured in minimal media to mimic starvation conditions such as that experienced by S. Typhimurium in the human intestinal tract, and the pathogen's virulence in vitro and in vivo was measured. S. Typhimurium cultured in minimal media displayed a reduced ability to invade human epithelial cells in a manner that was at least partially independent of the Salmonella Pathogenicity Island 1 (SPI-1) type III secretion system. Nutrient deprivation did not, however, alter the ability of S. Typhimurium to replicate and survive inside epithelial cells. In a murine model of S. Typhimurium-induced gastroenteritis, prior cultivation in minimal media did not alter the pathogen's ability to colonize mice, nor did it affect levels of gastrointestinal inflammation. Upon examining the post-infection fecal gastrointestinal microbiota, we found that specifically in the 129Sv/ImJ murine strain S. Typhimurium cultured in minimal media induced differential microbiota compositional shifts compared to that of S. Typhimurium cultured in rich media. Together these findings demonstrate that S. Typhimurium remains a potent pathogen even in the face of nutritional deprivation, but nevertheless that nutrient deprivation encountered in this environment elicits significant changes in the bacterium genetic programme, as well as its capacity to alter host microbiota composition.

Early infancy microbial and metabolic alterations affect risk of childhood asthma.

Asthma is the most prevalent pediatric chronic disease and affects more than 300 million people worldwide. Recent evidence in mice has identified a "critical window" early in life where gut microbial changes (dysbiosis) are most influential in experimental asthma. However, current research has yet to establish whether these changes precede or are involved in human asthma. We compared the gut microbiota of 319 subjects enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study, and show that infants at risk of asthma exhibited transient gut microbial dysbiosis during the first 100 days of life. The relative abundance of the bacterial genera Lachnospira, Veillonella, Faecalibacterium, and Rothia was significantly decreased in children at risk of asthma. This reduction in bacterial taxa was accompanied by reduced levels of fecal acetate and dysregulation of enterohepatic metabolites. Inoculation of germ-free mice with these four bacterial taxa ameliorated airway inflammation in their adult progeny, demonstrating a causal role of these bacterial taxa in averting asthma development. These results enhance the potential for future microbe-based diagnostics and therapies, potentially in the form of probiotics, to prevent the development of asthma and other related allergic diseases in children.

Gastrointestinal microbiota-mediated control of enteric pathogens.

The gastrointestinal (GI) microbiota is a complex community of microorganisms residing within the mammalian gastrointestinal tract. The GI microbiota is vital to the development of the host immune system and plays a crucial role in human health and disease. The composition of the GI microbiota differs immensely among individuals yet specific shifts in composition and diversity have been linked to inflammatory bowel disease, obesity, atopy, and susceptibility to infection. In this review, we describe the GI microbiota and its role in enteric diseases caused by pathogenic Escherichia coli, Salmonella enterica, and Clostridium difficile. We discuss the central role of the GI microbiota in protective immunity, resistance to enteric pathogens, and resolution of enteric colitis.

Antivirulence activity of the human gut metabolome.

The mammalian gut contains a complex assembly of commensal microbes termed microbiota. Although much has been learned about the role of these microbes in health, the mechanisms underlying these functions are ill defined. We have recently shown that the mammalian gut contains thousands of small molecules, most of which are currently unidentified. Therefore, we hypothesized that these molecules function as chemical cues used by hosts and microbes during their interactions in health and disease. Thus, a search was initiated to identify molecules produced by the microbiota that are sensed by pathogens. We found that a secreted molecule produced by clostridia acts as a strong repressor of Salmonella virulence, obliterating expression of the Salmonella pathogenicity island 1 as well as host cell invasion. It has been known for decades that the microbiota protects its hosts from invading pathogens, and these data suggest that chemical sensing may be involved in this phenomenon. Further investigations should reveal the exact biological role of this molecule as well as its therapeutic potential. Importance: Microbes can communicate through the production and sensing of small molecules. Within the complex ecosystem formed by commensal microbes living in and on the human body, it is likely that these molecular messages are used extensively during the interactions between different microbial species as well as with host cells. Deciphering such a molecular dialect will be fundamental to our understanding of host-microbe interactions in health and disease and may prove useful for the design of new therapeutic strategies that target these mechanisms of communication.

Effects of antibiotics on human microbiota and subsequent disease.

Although antibiotics have significantly improved human health and life expectancy, their disruption of the existing microbiota has been linked to significant side effects such as antibiotic-associated diarrhea, pseudomembranous colitis, and increased susceptibility to subsequent disease. By using antibiotics to break colonization resistance against Clostridium, Salmonella, and Citrobacter species, researchers are now exploring mechanisms for microbiota-mediated modulation against pathogenic infection, revealing potential roles for different phyla and family members as well as microbiota-liberated sugars, hormones, and short-chain fatty acids in regulating pathogenicity. Furthermore, connections are now being made between microbiota dysbiosis and a variety of different diseases such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, atopy, and obesity. Future advances in the rapidly developing field of microbial bioinformatics will enable researchers to further characterize the mechanisms of microbiota modulation of disease and potentially identify novel therapeutics against disease.

AglQ is a novel component of the Haloferax volcanii N-glycosylation pathway.

N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii 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 Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii 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 Hfx. volcanii N-glycosylation.

Two distinct N-glycosylation pathways process the Haloferax volcanii S-layer glycoprotein upon changes in environmental salinity.

UNLABELLED: N-glycosylation in Archaea presents aspects of this posttranslational modification not seen in either Eukarya or Bacteria. In the haloarchaeon Haloferax volcanii, the surface (S)-layer glycoprotein can be simultaneously modified by two different N-glycans. Asn-13 and Asn-83 are modified by a pentasaccharide, whereas Asn-498 is modified by a tetrasaccharide of distinct composition, with N-glycosylation at this position being related to environmental conditions. Specifically, N-glycosylation of Asn-498 is detected when cells are grown in the presence of 1.75 but not 3.4 M NaCl. While deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498, deletion of genes within the cluster spanning HVO_2046 to HVO_2061 interfered with the assembly and attachment of the Asn-498-linked tetrasaccharide. Transfer of the "low-salt" tetrasaccharide from the dolichol phosphate carrier upon which it is assembled to S-layer glycoprotein Asn-498 did not require AglB, the oligosaccharyltransferase responsible for pentasaccharide attachment to Asn-13 and Asn-83. Finally, although biogenesis of the low-salt tetrasaccharide is barely discernible upon growth at the elevated salinity, this glycan was readily detected under such conditions in strains deleted of pentasaccharide biosynthesis pathway genes, indicative of cross talk between the two N-glycosylation pathways. IMPORTANCE: In the haloarchaeon Haloferax volcanii, originally from the Dead Sea, the pathway responsible for the assembly and attachment of a pentasaccharide to the S-layer glycoprotein, a well-studied glycoprotein in this species, has been described. More recently, it was shown that in response to growth in low salinity, the same glycoprotein is modified by a novel tetrasaccharide. In the present study, numerous components of the pathway used to synthesize this "low-salt" tetrasaccharide are described. As such, this represents the first report of two N-glycosylation pathways able to simultaneously modify a single protein as a function of environmental salinity. Moreover, and to the best of our knowledge, the ability to N-glycosylate the same protein with different and unrelated glycans has not been observed in either Eukarya or Bacteria or indeed beyond the halophilic archaea, for which similar dual modification of the Halobacterium salinarum S-layer glycoprotein was reported.

AglS, a novel component of the Haloferax volcanii N-glycosylation pathway, is a dolichol phosphate-mannose mannosyltransferase.

In Haloferax volcanii, a series of Agl proteins mediates protein N-glycosylation. The genes encoding all but one of the Agl proteins are sequestered into a single gene island. The same region of the genome includes sequences also suspected but not yet verified as serving N-glycosylation roles, such as HVO_1526. In the following, HVO_1526, renamed AglS, is shown to be necessary for the addition of the final mannose subunit of the pentasaccharide N-linked to the surface (S)-layer glycoprotein, a convenient reporter of N-glycosylation in Hfx. volcanii. Relying on bioinformatics, topological analysis, gene deletion, mass spectrometry, and biochemical assays, AglS was shown to act as a dolichol phosphate-mannose mannosyltransferase, mediating the transfer of mannose from dolichol phosphate to the tetrasaccharide corresponding to the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein.

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

AglP is a S-adenosyl-L-methionine-dependent methyltransferase that participates in the N-glycosylation pathway of Haloferax volcanii.

While pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methionine-dependent methyltransferase.

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.

AglF, aglG and aglI, novel members of a gene island involved in the N-glycosylation of the Haloferax volcanii S-layer glycoprotein.

Proteins in all three domains of life can experience N-glycosylation. The steps involved in the archaeal version of this post-translational modification remain largely unknown. Hence, as the next step in ongoing efforts to identify components of the N-glycosylation pathway of the halophilic archaeon Haloferax volcanii, the involvement of three additional gene products in the biosynthesis of the pentasaccharide decorating the S-layer glycoprotein was demonstrated. The genes encoding AglF, AglI and AglG are found immediately upstream of the gene encoding the archaeal oligosaccharide transferase, AglB. Evidence showing that AglF and AglI are involved in the addition of the hexuronic acid found at position three of the pentasaccharide is provided, while AglG is shown to contribute to the addition of the hexuronic acid found at position two. Given their proximities in the H. volcanii genome, the transcription profiles of aglF, aglI, aglG and aglB were considered. While only aglF and aglI share a common promoter, transcription of the four genes is co-ordinated, as revealed by determining transcript levels in H. volcanii cells raised in different growth conditions. Such changes in N-glycosylation gene transcription levels offer additional support for the adaptive role of this post-translational modification in H. volcanii.

Sweet to the extreme: protein glycosylation in Archaea.

Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.

Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer.

In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.