Host-bacterial Interactions Research Articles

Translational Fidelity during Bacterial Stresses and Host Interactions.

Translational fidelity refers to accuracy during protein synthesis and is maintained in all three domains of life. Translational errors occur at base levels during normal conditions and may rise due to mutations or stress conditions. In this article, we review our current understanding of how translational fidelity is perturbed by various environmental stresses that bacterial pathogens encounter during host interactions. We discuss how oxidative stress, metabolic stresses, and antibiotics affect various types of translational errors and the resulting effects on stress adaption and fitness. We also discuss the roles of translational fidelity during pathogen-host interactions and the underlying mechanisms. Many of the studies covered in this review will be based on work with Salmonella enterica and Escherichia coli, but other bacterial pathogens will also be discussed.

The Local Activation of Toll-like Receptor 7 (TLR7) Modulates Colonic Epithelial Barrier Function in Rats.

Toll-like receptors (TLRs)-mediated host-bacterial interactions participate in the microbial regulation of gastrointestinal functions, including the epithelial barrier function (EBF). We evaluated the effects of TLR7 stimulation on the colonic EBF in rats. TLR7 was stimulated with the selective agonist imiquimod (100/300 µg/rat, intracolonic), with or without the intracolonic administration of dimethyl sulfoxide (DMSO). Colonic EBF was assessed in vitro (electrophysiology and permeability to macromolecules, Ussing chamber) and in vivo (passage of macromolecules to blood and urine). Changes in the expression (RT-qPCR) and distribution (immunohistochemistry) of tight junction-related proteins were determined. Expression of proglucagon, precursor of the barrier-enhancer factor glucagon-like peptide 2 (GLP-2) was also assessed (RT-qPCR). Intracolonic imiquimod enhanced the EBF in vitro, reducing the epithelial conductance and the passage of macromolecules, thus indicating a pro-barrier effect of TLR7. However, the combination of TLR7 stimulation and DMSO had a detrimental effect on the EBF, which manifested as an increased passage of macromolecules. DMSO alone had no effect. The modulation of the EBF (imiquimod alone or with DMSO) was not associated with changes in gene expression or the epithelial distribution of the main tight junction-related proteins (occludin, tricellulin, claudin-2, claudin-3, junctional adhesion molecule 1 and Zonula occludens-1). No changes in the proglucagon expression were observed. These results show that TLR7 stimulation leads to the modulation of the colonic EBF, having beneficial or detrimental effects depending upon the state of the epithelium. The underlying mechanisms remain elusive, but seem independent of the modulation of the main tight junction-related proteins or the barrier-enhancer factor GLP-2.

The impact of C. elegans ceramide glucosyltransferase enzymes on the beneficial effects of B. subtilis lifespan.

Ceramide glucosyltransferase (CGT) adds sugar moieties to ceramide, forming glucosylceramides that play roles in immune signaling, stress response, and host-bacterial interactions. Here, we examined whether mutations in cgt block the beneficial effects of Bacillus subtilis on C. elegans lifespan. We found that loss of cgt-1 or cgt-3 reduces lifespan compared to wildtype worms, but did not prevent the lifespan-extending phenotype of B. subtilis . However, cgt-1(ok1045) and cgt-3(tm504) did play a minor role in blocking stress resistance of 5-day old worms treated with B. subtilis . Further studying CGTs may elucidate potential roles of glucosylceramides in host-bacterial interaction.

Effects of Phycosphere Bacteria on Their Algal Host Are Host Species-Specific and Not Phylogenetically Conserved.

Phytoplankton is fundamental to life on Earth. Their productivity is influenced by the microbial communities residing in the phycosphere surrounding algal cells. Expanding our knowledge on how algal-bacterial interactions affect algal growth to more hosts and bacteria can help elucidate general principles of algal-host interactions. Here, we isolated 368 bacterial strains from phycosphere communities, right after phycosphere recruitment from pond water and after a month of lab cultivation and examined their impacts on growth of five green algal species. We isolated both abundant and rare phycosphere members, representing 18.4% of the source communities. Positive and neutral effects predominated over negative effects on host growth. The proportion of each effect type and whether the day of isolation mattered varied by host species. Bacteria affected algal carrying capacity more than growth rate, suggesting that nutrient remineralization and toxic byproduct metabolism may be a dominant mechanism. Across-host algal fitness assays indicated host-specific growth effects of our isolates. We observed no phylogenetic conservation of the effect on host growth among bacterial isolates. Even isolates with the same ASV had divergent effects on host growth. Our results emphasize highly specific host-bacterial interactions in the phycosphere and raise questions as to which mechanisms mediate these interactions.

Contribution of Uncharacterized Target Genes of MxtR/ErdR to Carbon Source Utilization by Pseudomonas putida KT2440.

MxtR/ErdR is a two-component system that has been previously described as a regulator of the utilization of acetate in Vibrio cholerae and in some Pseudomonas species. Regulation is achieved by controlling the expression of the acs gene (acetyl-coenzyme A [CoA] synthetase). However, the physiological significance of other identified target genes is not fully understood. Here, we investigated the role of pp_0154 (scpC) and pp_0354/pp_0353 in the soil bacterium Pseudomonas putida KT2440. To this end, the genes were individually deleted and complemented in trans. Then, the growth of the resulting strains on different carbon sources was analyzed. To obtain information on protein function, a bioinformatic analysis was performed, and ScpC was purified and characterized in vitro. Our results indicated that scpC is important for P. putida KT2440 to cope with high concentrations of acetate. The encoded enzyme catalyzes the transfer of coenzyme A between acetate and succinate. On the contrary, pp_0353 and pp_0354 proved to be unimportant for the growth of the strain on acetate under our conditions. Extending the phenotypic analysis to other carbon sources led to the discovery that mxtR, erdR, and pp_0353 are important for the utilization of pyruvate as a carbon source. Taken together, the findings of this study expand the knowledge about the role of the MxtR/ErdR two-component system in carbon source utilization and about the specific functions of its target genes. IMPORTANCE MxtR/ErdR and homologous two-component systems play important roles in the regulatory networks that control cell metabolism and influence bacterial-host interactions. Using the MxtR/ErdR two-component system of the plant growth-promoting soil bacterium Pseudomonas putida KT2440 as a model, this work elucidates the function of previously uncharacterized target genes of MxtR/ErdR and extends the knowledge of the physiological significance of the two-component system. Our results suggest that the target gene scpC encodes an acetate:succinate CoA transferase that is involved in the detoxification of acetate when it is present in large amounts. Furthermore, it is shown that MxtR/ErdR controls the metabolism of not only acetate but also pyruvate. This control involves the target gene pp_0353 (putative exonuclease). These findings may facilitate the optimization of P. putida KT2440 as a chassis for biotechnological applications and may contribute to a better understanding of the regulatory network of pathogens like Pseudomonas aeruginosa.

Oral microbiomes of patients with infective endocarditis (IE): a comparative pilot study of IE patients, patients at risk for IE and healthy controls

ABSTRACT Background Infective endocarditis (IE) is an uncommon disease with high morbidity and mortality rates, which often develops from oral bacterial species entering circulation. Objective We compared oral microbiome profiles of three groups: IE patients (N 9 patients; n = 27 samples), disease controls at risk for IE (N = 28; n = 84), and healthy controls (N = 37; n = 111). Bacterial species in IE patients’ blood cultures were identified for comparison with matched oral samples. Design Oral microbiome profiles were obtained from buccal mucosa, saliva, and tongue samples for all three groups and from sub- and supra-gingival plaque samples of the IE group (N = 9; n = 16) and disease controls (N = 28; n = 54). Alpha- and beta-diversities were determined based on relative abundance data. Discriminative species were identified by LEfSe, post hoc Mann-Whitney, and ROC analyses. Identity of the bacterial species in IE patients’ blood cultures was confirmed by 16S-rRNA gene Sanger sequencing. Results Alpha- and beta-diversities differed between groups. Discriminative IE-associated species were identified, e.g. Haemophilus parainfluenzae and Streptococcus sanguinis. Two blood isolates were Staphylococcus aureus, also identified in one matched saliva sample. Streptococcus mutans was present in one patient’s plaque samples and blood culture. Conclusions Oral microbiomes of IE, non-IE disease controls, and healthy controls differed significantly. A better understanding of IE-related bacterial-host interactions is warranted.

Intra-Amniotic Administration-An Emerging Method to Investigate Necrotizing Enterocolitis, In Vivo (Gallus gallus).

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease in premature infants and a leading cause of death in neonates (1-7% in the US). NEC is caused by opportunistic bacteria, which cause gut dysbiosis and inflammation and ultimately result in intestinal necrosis. Previous studies have utilized the rodent and pig models to mimic NEC, whereas the current study uses the in vivo (Gallus gallus) intra-amniotic administration approach to investigate NEC. On incubation day 17, broiler chicken (Gallus gallus) viable embryos were injected intra-amniotically with 1 mL dextran sodium sulfate (DSS) in H2O. Four treatment groups (0.1%, 0.25%, 0.5%, and 0.75% DSS) and two controls (H2O/non-injected controls) were administered. We observed a significant increase in intestinal permeability and negative intestinal morphological changes, specifically, decreased villus surface area and goblet cell diameter in the 0.50% and 0.75% DSS groups. Furthermore, there was a significant increase in pathogenic bacterial (E. coli spp. and Klebsiella spp.) abundances in the 0.75% DSS group compared to the control groups, demonstrating cecal microbiota dysbiosis. These results demonstrate significant physiopathology of NEC and negative bacterial-host interactions within a premature gastrointestinal system. Our present study demonstrates a novel model of NEC through intra-amniotic administration to study the effects of NEC on intestinal functionality, morphology, and gut microbiota in vivo.

Recent advances on the regulation of bacterial biofilm formation by herbal medicines.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

Insights into the temperature responses of Pseudomonas species in beneficial and pathogenic host interactions.

Pseudomonas species are metabolically versatile bacteria able to exploit a wide range of ecological niches. Different Pseudomonas species can grow as free-living cells, biofilms, or associated with plants or animals, including humans, and their ecological success partially lies in their ability to grow and adapt to different temperatures. These bacteria are relevant for human activities, due to their clinical importance and their biotechnological potential for different applications such as bioremediation and the production of biopolymers, surfactants, secondary metabolites, and enzymes. In agriculture, some of them can act as plant growth promoters and are thus used as inoculants, whereas others, like P. syringae pathovars, can cause disease in commercial crops. This review aims to provide an overview of the temperature-response mechanisms in Pseudomonas species, looking for novel features or strategies based on techniques such as transcriptomics and proteomics. We focused on temperature-dependent traits mainly associated with virulence, host colonization, survival, and production of secondary metabolites. We analyzed human, animal, and plant pathogens and plant growth-promoting Pseudomonas species, including P. aeruginosa, P. plecoglossicida, several P. syringae pathovars, and P. protegens. Our aim was to provide a comprehensive view of the relevance of temperature-response traits in human and animal health and agricultural applications. Our analysis showed that features relevant to the bacterial-host interaction are adjusted to the environmental or host temperature regardless of the optimal growth temperature in the laboratory, and thus contribute to improving bacterial fitness. KEY POINTS: • In Pseudomonas species, temperature impacts the bacterial-host interaction. • Interaction traits are expressed at temperatures different from the optimal reported. • The bacterial-host interaction could be affected by climate change.

Extracellular vesicles, a novel model linking bacteria to ferroptosis in the future?

Ferroptosis is a recently discovered modulated cell death mechanism caused by the accumulation of iron-dependent lipid peroxides to toxic levels and plays an important role in tumor immunology and neurology. Recent studies have shown that ferroptosis may play a crucial role in bacterial infection pathogenesis, which may be useful in anti-infection therapies. However, how bacteria enter cells to induce ferroptosis after invading the host immune system remains largely unknown. In addition, the current studies only focus on the relationship between a single bacterial species or genus and host cell ferroptosis, and there is no systematic summary of its regulatory mechanism. Therefore, our review firstly sums up the role of ferroptosis in bacterial infection and its regulatory mechanism, and innovatively speculates on the function and potential mechanism of extracellular vesicles (EVs) in bacterial-induced ferroptosis, in order to provide possible novel directions and ideas for future anti-infection research. KEY POINTS: • Ferroptosis presents a novel mechanism for bacterial host interaction • EVs provide the potential mechanism for bacterial-induced ferroptosis • The relationship of EVs with ferroptosis provides possible directions for future treatment of bacterial infection.

Serum 3-phenyllactic acid level is reduced in benign multiple sclerosis and is associated with effector B cell ratios

Background3-phenyllactic acid (PLA) is produced by both intestinal bacteria and the human host. PLA exists in its D- and L- chiral forms. It modulates human immune functions, thereby acting as a mediator of bacterial-host interactions. We aim to determine the amount and potential influence of PLA on clinical and immunological features of MS. MethodsWe measured D- and L-PLA levels in bacterial supernatants and in sera of 60 MS patients and 25 healthy controls. We investigated potential associations between PLA levels, clinical features of MS, serum cytokine levels and ratios of peripheral blood lymphocyte subsets. ResultsMultiple gut commensal bacteria possessed the capacity to generate D- and L-PLA. MS patients with benign phenotype showed markedly lower PLA levels than healthy controls or other MS patients. Fingolimod resistant patients had higher PLA levels at baseline. Furthermore, MS patients with higher PLA levels tended to display increased memory B and plasma cell ratios, elevated IL-4 levels and increased ratios of IL-4 and IL-10 producing T cell subsets. ConclusionCollectively, our work indicates that reduced serum levels of PLA could be associated with a favorable clinical course in MS and possibly be used as a biomarker.

Overview of the causes and types of recurrent cystitis

Bacterial cystitis accounts for the majority of urinary tract infection (UTI). It is frequently found in young, otherwise healthy females showing no signs of anatomical or physiological urinary tract defects. Among young women who have experienced an episode of acute bacterial cystitis, 25% to 50% develop recurrent episodes. Individuals with recurrent cystitis episodes have a greater susceptibility to urogenital colonization with uropathogens that thrive by adhering to uroepithelial cells. Further, there is an increased likelihood of infection with an antibiotic resistant uropathogen due to previous antimicrobial therapy for recurring infections. At each step of the pathogenesis, different host genetic, biologic, and behavioral factors, and bacterial factors interact and influence susceptibility to recurrent cystitis. Behavioral factors like sexual intercourse, use of spermicidal contraceptives and history of recurrent cystitis are major independent risk factors for recurrent cystitis infections. In post-menopausal women without comorbidities, estrogen depletion is mainly responsible for increased susceptibility to recurrent cystitis. Certain strain-specific bacterial virulence determinants may also contribute to recurrent cystitis by providing a selective advantage. Phenotypically, recurrent cystitis is categorized as reinfection or bacterial persistence. Majority of recurrent cystitis cases seen in men occur due to structural or functional abnormalities of the urinary bladder which allows same pathogens from the same site in the bladder to cause recurrences due to bacterial persistence. The concept of ‘uncomplicated’ and ‘complicated’ recurrent cystitis is used for classification of recurrent cystitis cases, similar to UTIs. Uncomplicated recurrent cystitis comprises cases occurring in young, healthy, nonpregnant women, mainly due to uropathogenic Escherichia coli. Due to the unique spectrum of host-bacterial interactions in the urinary tract, the distinction between complicated and uncomplicated recurrent cystitis is not straightforward. ‘Complicating’ factors such as structural, obstructive, neoplastic, functional, and neurological abnormalities of the urinary tract, systemic conditions including pregnancy, and certain demographic factors are attributed in the development of complicated recurrent bacterial cystitis.

Interactions of the bacteriome, virome, and immune system in the nose.

Emerging evidence suggests that the nasal microbiome may influence host susceptibility to initial development and severity of respiratory viral infections. While not as extensively studied as the microbiota of the alimentary tract, it is now clearly established that the microbial composition of this niche is influenced by medical, social and pharmacological influences, predisposing some sub-populations to respiratory infections. The resulting specific microbial profiles may explain variance in susceptibility to viral infection. This review summaries the evolution and constituents of the commensal nasal microbiome; the bacterial-virus, bacterial-host and interbacterial interactions which potentiate disease; and considers the effects of interventions such as vaccination and probiotics.

Host-Bacterial Interactions: Outcomes of Antimicrobial Peptide Applications.

The bacterial membrane is part of a secretion system which plays an integral role to secrete proteins responsible for cell viability and pathogenicity; pathogenic bacteria, for example, secrete virulence factors and other membrane-associated proteins to invade the host cells through various types of secretion systems (Type I to Type IX). The bacterial membrane can also mediate microbial communities’ communication through quorum sensing (QS), by secreting auto-stimulants to coordinate gene expression. QS plays an important role in regulating various physiological processes, including bacterial biofilm formation while providing increased virulence, subsequently leading to antimicrobial resistance. Multi-drug resistant (MDR) bacteria have emerged as a threat to global health, and various strategies targeting QS and biofilm formation have been explored by researchers worldwide. Since the bacterial secretion systems play such a crucial role in host–bacterial interactions, this review intends to outline current understanding of bacterial membrane systems, which may provide new insights for designing approaches aimed at antimicrobials discovery. Various mechanisms pertaining interaction of the bacterial membrane with host cells and antimicrobial agents will be highlighted, as well as the evolution of bacterial membranes in evasion of antimicrobial agents. Finally, the use of antimicrobial peptides (AMPs) as a cellular device for bacterial secretion systems will be discussed as emerging potential candidates for the treatment of multidrug resistance infections.

The Intra-amniotic Administration- an Emerging Method to Investigate Necrotizing Enterocolitis, In Vivo (Gallus gallus)

ObjectivesDemonstrate necrotizing enterocolitis (NEC) in a novel in vivo (Gallus gallus) model through intra-amniotic administration of dextran sodium sulfate (DSS, demonstrated agent to induce NEC), to induce NEC pathology, pathophysiology, and negative bacterial-host interactions within a premature gastrointestinal system.MethodsGallus gallus embryos were injected intra-amniotically with 1 mL DSS in DI H2O. Four treatment groups (0.1%, 0.25%, 0.5%, and 0.75% DSS) and two controls (H2O/non-injected controls) were administered. Upon hatch, blood, cecum, small intestine, and liver were collected to assess hemoglobin and intestinal permeability, intestinal microbiota alterations, intestinal morphometric assessment, and mRNA gene expression of relevant key nutrient transporters/enzymes inflammatory proteins, respectively.ResultsResults indicated that intestinal permeability was significantly increased post DSS exposure, and negative intestinal morphological changes were found. In the 0.50% and 0.75% DSS groups, villus surface area and goblet cell diameter have significantly decreased (p < 0.05). Furthermore, there was a significant (p < 0.05) increase in pathogenic bacterial abundance (E. coli and Klebsiella) in the 0.75% DSS group compared to the controls, demonstrating cecal microbiota dysbiosis. Taken together, these results demonstrate striking similarities between current observations in the Gallus gallus model compared with NEC patients.ConclusionsThis study is the first to demonstrate NEC symptoms through intra-amniotic administration of DSS in vivo (Gallus gallus), whereas previous studies have utilized rodent and pig models. The results of this study are promising evidence to investigate increased concentrations of DSS further to cause more severe NEC symptoms and identify potential novel biomarkers of less severe NEC cases. The development of such a model also allows the assessment of potential dietary bioactives that may ameliorate the effects of NEC.Funding SourcesNo external funding was utilized for the study.

Diversity and distribution of Type VI Secretion System gene clusters in bacterial plasmids

Type VI Secretion System (T6SS) is a nanomolecular apparatus that allows the delivery of effector molecules through the cell envelope of a donor bacterium to prokaryotic and/or eukaryotic cells, playing a role in the bacterial competition, virulence, and host interaction. T6SS is patchily distributed in bacterial genomes, suggesting an association with horizontal gene transfer (HGT). In fact, T6SS gene loci are eventually found within genomic islands (GIs), and there are some reports in plasmids and integrative and conjugative elements (ICEs). The impact that T6SS may have on bacteria fitness and the lack of evidence on its spread mechanism led us to question whether plasmids could represent a key mechanism in the spread of T6SS in bacteria. Therefore, we performed an in-silico analysis to reveal the association between T6SS and plasmids. T6SS was mined on 30,660 plasmids from NCBI based on the presence of at least six T6SS core proteins. T6SS was identified in 330 plasmids, all belonging to the same type (T6SSi), mainly in Proteobacteria (328/330), particularly in Rhizobium and Ralstonia. Interestingly, most genomes carrying T6SS-harboring plasmids did not encode T6SS in their chromosomes, and, in general, chromosomal and plasmid T6SSs did not form separate clades.

Identification and Characterization of Small Regulatory RNA in Streptococcus

The human gut is a rich habitat for a diverse population of bacteria. These bacteria play a vital role in a multitude of functions by interacting with each‐other and the human host. They can play roles in immune responses, metabolism, and even neurological activities. Some inhabitants of the gut biome include the lactic acid bacteria: Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. These bacteria are essential members of the flora and are non‐pathogenic, however, some of their relatives like Streptococcus pyogenes cause severe diseases like toxic shock syndrome and flesh‐eating disease. The metabolic functions of these bacteria are mediated by small regulatory RNAs (sRNAs), which are noncoding RNA sequences that fold into complex stem‐loop structures. The goal of this project was to identify and characterize the small regulatory RNAs that mediate bacterial metabolism and host interactions in non‐pathogenic, lactic acid bacteria. A small regulatory RNA that is conserved among different streptococcal species was identified. In S. pyogenes, this conserved sRNA, named MarS, is associated with virulence, however, the function of its ortholog found in non‐pathogenic bacteria, AsdS sRNA, has not been well‐characterized. Computational methods were used to elucidate the function of this sRNA as well as predict its 2D and 3D structures. The gene containing the target sRNA was isolated and RNA constructs were designed to characterize regions that are part of it. Future work will be focused on structure determination and characterizing interactions to target sRNA through biophysical and biochemical methods.

Leveraging Antibody, B Cell and Fc Receptor Interactions to Understand Heterogeneous Immune Responses in Tuberculosis

Despite over a century of research, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), continues to kill 1.5 million people annually. Though less than 10% of infected individuals develop active disease, the specific host immune responses that lead to Mtb transmission and death, as well as those that are protective, are not yet fully defined. Recent immune correlative studies demonstrate that the spectrum of infection and disease is more heterogenous than has been classically defined. Moreover, emerging translational and animal model data attribute a diverse immune repertoire to TB outcomes. Thus, protective and detrimental immune responses to Mtb likely encompass a framework that is broader than T helper type 1 (Th1) immunity. Antibodies, Fc receptor interactions and B cells are underexplored host responses to Mtb. Poised at the interface of initial bacterial host interactions and in granulomatous lesions, antibodies and Fc receptors expressed on macrophages, neutrophils, dendritic cells, natural killer cells, T and B cells have the potential to influence local and systemic adaptive immune responses. Broadening the paradigm of protective immunity will offer new paths to improve diagnostics and vaccines to reduce the morbidity and mortality of TB.

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility.

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

In the Thick of It: Formation of the Tuberculous Granuloma and Its Effects on Host and Therapeutic Responses

The defining pathology of tuberculosis is the granuloma, an organized structure derived from host immune cells that surrounds infecting Mycobacterium tuberculosis. As the location of much of the bacteria in the infected host, the granuloma is a central point of interaction between the host and the infecting bacterium. This review describes the signals and cellular reprogramming that drive granuloma formation. Further, as a central point of host-bacterial interactions, the granuloma shapes disease outcome by altering host immune responses and bacterial susceptibility to antibiotic treatment, as discussed herein. This new understanding of granuloma biology and the signaling behind it highlights the potential for host-directed therapies targeting the granuloma to enhance antibiotic access and tuberculosis-specific immune responses.