Virulence Associated Genes-Deleted Salmonella Montevideo is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens

To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated and highly immunogenic strains, JOL1625 (∆lon), JOL1597 (∆cpxR), and JOL1599 (∆lon∆cpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in extracellular polysaccharide (EPS) production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in faeces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that ∆lon- and/or ∆cpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections

Host Mucin Is Exploited by Pseudomonas aeruginosa To Provide Monosaccharides Required for a Successful Infection

One of the first lines of defense present at mucosal epithelial tissues is mucus, which is a highly viscous material formed by mucin glycoproteins. Mucins serve various functions, but importantly they aid in the clearance of pathogens and debris from epithelial barriers and serve as innate immune factors. In this study, we describe a requirement of host monosaccharides, likely derived from host mucins, for the ability of Pseudomonas aeruginosa to colonize the intestine and ultimately cause death in Caenorhabditis elegans. We also demonstrate that monosaccharides alter the ability of bacteria to bind to both Caenorhabditis elegans intestinal cells and human lung alveolar epithelial cells, suggesting that there are conserved mechanisms underlying host-pathogen interactions in a range of organisms. By gaining a better understanding of pathogen-mucin interactions, we can develop better approaches to protect against pathogen infection.One of the primary functions of the mucosal barrier, found lining epithelial cells, is to serve as a first-line of defense against microbial pathogens. The major structural components of mucus are heavily glycosylated proteins called mucins. Mucins are key components of the innate immune system as they aid in the clearance of pathogens and can decrease pathogen virulence. It has also been recently reported that individual mucins and derived glycans can attenuate the virulence of the human pathogen Pseudomonas aeruginosa. Here, we show data indicating that mucins not only play a role in host defense but that they can also be subverted by P. aeruginosa to cause disease. We found that the mucin MUL-1 and mucin-derived monosaccharides N-acetyl-galactosamine and N-acetylglucosamine are required for P. aeruginosa killing of Caenorhabditis elegans. We also found that the defective adhesion of P. aeruginosa to human lung alveolar epithelial cells, deficient in the mucin MUC1, can be reversed by the addition of individual monosaccharides. The monosaccharides identified in this study are found in a wide range of organisms where they act as host factors required for bacterial pathogenesis. While mucins in C. elegans lack sialic acid caps, which makes their monosaccharides readily available, they are capped in other species. Pathogens such as P. aeruginosa that lack sialidases may rely on enzymes from other bacteria to utilize mucin-derived monosaccharides

Additional file 4 of Dissection of a sensorimotor circuit underlying pathogen aversion in C. elegans

Additional file 4: Fig. S4. AUA and RMG neurons are required for motor neuron oscillations upon AWB and command interneuron stimulation in simulations of the C. elegans nervous system. (A) The same data as in Fig. 3A and B presented as waveforms. (B) The same data as in Fig. 3D presented as waveforms. (C) The same data as in Fig. 3E presented as waveforms

Additional file 2 of Dissection of a sensorimotor circuit underlying pathogen aversion in C. elegans

Additional file 2: Fig. S2. AWC, ASI, and ASE neurons do not lead to oscillations in motor neurons important for backward locomotion. Heatmaps (above) and waveforms (below) of VD, DD, VA, DA, and AS motor neuron activity in the Neural Interactome upon 0.9 nA stimulation of the CIs and 5.0 nA stimulation of AWC (A), ASI (B), and ASE (C)

Additional file 7 of Dissection of a sensorimotor circuit underlying pathogen aversion in C. elegans

Additional file 7: Fig. S7. Correlation of intestinal distention and learned reflexive aversion for P. aeruginosa but not E. faecalis exposure. (A) PA14::GFP relative fluorescence in the intestine (x-axis) and the trained response index to P. aeruginosa (y-axis) were measured in individual animals (dots), and linear regression was performed (red line). (B) Same as A but with intestinal diameter on P. aeruginosa (x-axis). (C) Same as A but with E. faecalis. (D) Same as B but with E. faecalis. R2 and P-values are shown next to linear regression lines in red

Additional file 5 of Dissection of a sensorimotor circuit underlying pathogen aversion in C. elegans

Additional file 5: Fig. S5. AIB, AVB, or SMB neuron ablation does not diminish oscillations in backward locomotion-associated motor neurons. Heatmaps (left) and waveforms (right) of activity of motor neurons (rows) upon 5.0 nA stimulation of AWB neurons and 0.9 nA stimulation of the CIs with AIB (A), AVB (B), or SMB (C) neurons ablated in the Neural Interactome. (D) Response index to P. aeruginosa for both naïve (gray) and trained (green) animals with either no neurons ablated (N2, WT) or AIB (JN578) or AVB (ZM7297) neurons ablated. For ZM7297 animals, the miniSOG ablation protocol was followed as in Fig. 2A. Two-way ANOVA with subsequent comparison to naïve or trained WT groups was performed. Error bars depict standard deviation. N = 25 (individual dots) for all groups

Additional file 6 of Dissection of a sensorimotor circuit underlying pathogen aversion in C. elegans

Additional file 6: Fig. S6. Genetic ablation of AUA and RMG neurons. (A) Representative fluorescent micrographs of NY2078 ynIs78 [flp-8p::GFP] (left) and AY178 ynIs78 [flp-8p::GFP]; flp-8p::ced-3 (p15)::nz + flp-32::cz::ced-3 (p17) + unc-122p::rfp (right) animals. (B) Representative fluorescent micrographs of NY2087 ynIs87 [flp-21p::GFP (left) and AY179 ynIs87 [flp-21p::GFP]; flp-21p::ced-3 (p15)::nz + ncs-1p::cz::ced-3 (p17) + unc-122p::rfp (right) animals. White, filled arrows point to intact AUA or RMG neurons, while white, unfilled arrows point to the lack of AUA or RMG neurons. GFP-positive neurons were counted for both intact and ablated animals (pink circles), showing that only the targeted neurons were ablated, with other neurons left intact. AUA intact: 4 GFP neurons; AUA ablated: 2 GFP neurons; RMG intact: 15 GFP neurons; RMG ablated: 13 neurons. Scale bars are 50 μm