Comparing the whole genome methylation landscape of dairy calf blood cells revealed intergenerational inheritance of the maternal metabolism.

This study evaluated the hypothesis that the maternal metabolic stressed status could be inherited to their F1 daughters via epigenetic mechanism. The maternal cow blood ß-hydroxybutyric acid (BHB) level (≥0.9 mM/L) was used as an indicator of maternal metabolic stress. Eight newborn daughters' blood cells were used for methylation comparison and analysis. By Whole Genome Bisulphite Sequencing (WGBS), a total of 1,861 Differentially Methylated Regions (DMRs), including 944 differentially methylated cytosines (DMCs), were identified. Most DMRs were distributed in intronic and intergenic regions, and most of the DMR in promoter regions were hypermethylated. Differentially methylated genes (DMGs) with DMR methylation differences higher than 20% were mainly enriched in metabolism-related pathways. These results suggest that newborn calves' metabolic pathways were altered, with 64 DMGs being clustered with metabolic signalling by KEGG analysis. Our study revealed the whole epigenetic landscape of calf blood cells and suggested that the maternal metabolic status can affect the embryo's epigenetic status and metabolic-related pathways in offspring, providing further evidence for epigenetic intergenerational inheritance of metabolic stress in domestic animals. Besides, this study also contributed more evidence to support the Developmental Origins of Health and Disease (DOHAD) theory in large animals.

Antibiotic treatment-induced secondary IgA deficiency enhances susceptibility to Pseudomonas aeruginosa pneumonia.

Broad-spectrum antibiotics are widely used with patients in intensive care units (ICUs), many of whom develop hospital-acquired infections with Pseudomonas aeruginosa. Although preceding antimicrobial therapy is known as a major risk factor for P. aeruginosa-induced pneumonia, the underlying mechanisms remain incompletely understood. Here we demonstrate that depletion of the resident microbiota by broad-spectrum antibiotic treatment inhibited TLR-dependent production of a proliferation-inducing ligand (APRIL), resulting in a secondary IgA deficiency in the lung in mice and human ICU patients. Microbiota-dependent local IgA contributed to early antibacterial defense against P. aeruginosa. Consequently, P. aeruginosa-binding IgA purified from lamina propria culture or IgA hybridomas enhanced resistance of antibiotic-treated mice to P. aeruginosa infection after transnasal substitute. Our study provides a mechanistic explanation for the well-documented risk of P. aeruginosa infection following antimicrobial therapy, and we propose local administration of IgA as a novel prophylactic strategy.

Spectrum of pathogen- and model-specific histopathologies in mouse models of acute pneumonia.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.

NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence.

Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1ß and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1ß and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes.

Anoxia and glucose supplementation preserve neutrophil viability and function.

Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 µg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.

Role of the N-Acetylmuramoyl-l-Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence.

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.

NOD-Like Receptors in Lung Diseases.

The lung is a particularly vulnerable organ at the interface of the body and the exterior environment. It is constantly exposed to microbes and particles by inhalation. The innate immune system needs to react promptly and adequately to potential dangers posed by these microbes and particles, while at the same time avoiding extensive tissue damage. Nucleotide-binding oligomerization domain-like receptors (NLRs) represent a group of key sensors for microbes and damage in the lung. As such they are important players in various infectious as well as acute and chronic sterile inflammatory diseases, such as pneumonia, chronic obstructive pulmonary disease (COPD), acute lung injury/acute respiratory distress syndrome, pneumoconiosis, and asthma. Activation of most known NLRs leads to the production and release of pro-inflammatory cytokines, and/or to the induction of cell death. We will review NLR functions in the lung during infection and sterile inflammation.

O-antigen protects gram-negative bacteria from histone killing.

Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous Escherichia coli libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens Shigella flexneri and Klebsiella pneumoniae.

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria.

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram-negative pathogen Salmonella typhimurium and the Gram-positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.

Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori.

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

Peptidoglycan detection by mammals and flies.

Peptidoglycan is an essential component of bacteria. The host exploits the peptidoglycan particular composition and uniqueness to bacteria for specific bacterial recognition. Insects and mammals accomplish this via receptors such as PGRP and Nod proteins.

Characterization of Helicobacter pylori lytic transglycosylases Slt and MltD.

Peptidoglycan (PG) is a cell wall heteropolymer that is essential for cell integrity. PG hydrolases participate in correct assembly of the PG layer and have been shown to be required for cell division, cell daughter separation, and maintenance of bacterial morphology. In silico analysis of the Helicobacter pylori genome resulted in identification of three potential hydrolases, Slt, MltD, and AmiA. This study was aimed at determining the roles of the putative lytic transglycosylases, Slt and MltD, in H. pylori morphology, growth, and PG metabolism. Strain 26695 single mutants were constructed using a nonpolar kanamycin cassette. The slt and mltD mutants formed normal bacillary and coccoid bacteria in the exponential and stationary phases, respectively. The slt and mltD mutants had growth rates comparable to the growth rate of the parental strain. However, the mltD mutant exhibited enhanced survival in the stationary phase compared to the wild type or the slt mutant. PG was purified from exponentially growing bacteria and from bacteria in the stationary phase, and its muropeptide composition was analyzed by high-pressure liquid chromatography. This analysis revealed changes in the muropeptide composition indicating that MltD and Slt have lytic transglycosylase activities. Glycan strand analysis suggested that Slt and MltD have exo and endo types of lytic transglycosylase activity, indicating that Slt is involved mainly in PG turnover and MltD is involved mainly in rearrangement of the PG layer. In this study, we determined the distinct roles of the lytic transglycosylases Slt and MltD in PG metabolism.

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape.

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-beta(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-kappaB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.

Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway.

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.

Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island.

Epithelial cells can respond to conserved bacterial products that are internalized after either bacterial invasion or liposome treatment of cells. We report here that the noninvasive Gram-negative pathogen Helicobacter pylori was recognized by epithelial cells via Nod1, an intracellular pathogen-recognition molecule with specificity for Gram-negative peptidoglycan. Nod1 detection of H. pylori depended on the delivery of peptidoglycan to host cells by a bacterial type IV secretion system, encoded by the H. pylori cag pathogenicity island. Consistent with involvement of Nod1 in host defense, Nod1-deficient mice were more susceptible to infection by cag pathogenicity island-positive H. pylori than were wild-type mice. We propose that sensing of H. pylori by Nod1 represents a model for host recognition of noninvasive pathogens.