Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Dual role of commensal bacteria in viral infections.

With our abilities to culture and sequence the commensal bacteria that dwell on and within a host, we can now study the host in its entirety, as a supraorganism that must be navigated by the pathogen invader. At present, the majority of studies have focused on the interaction between the host's microbiota and bacterial pathogens. This is not unwarranted, given that bacterial pathogens must compete with commensal organisms for the limited territory afforded by the host. However, viral pathogens also enter the host through surfaces coated with microbial life and encounter an immune system shaped by this symbiotic community. Therefore, we believe that the microbiota cannot be ignored when examining the interplay between the host and viral pathogens. Here, we review work that details mechanisms by which the microbiota either promotes or inhibits viral replication and virally induced pathogenesis. The impact of the microbitota on viral infection promises to be a new and exciting avenue of investigation, which will ultimately lead to better treatments and preventions of virally induced disease.

Antibody-mediated immune control of a retrovirus does not require the microbiota.

Commensal microbes are often required to control viral infection by facilitating host immune defenses. However, we found that this does not hold true for retroviral infection. We report that retrovirus-resistant mice control the pathogen with virus-neutralizing antibodies independently of commensal microbiota. This is in contrast to orthomyxoviruses and arenaviruses, where resistance is ablated in animals depleted of microbiota. Clearly, when it comes to antiviral immunity, the role of the microbiota cannot be generalized.

Exploring Alcohol-Related Behaviours in an Adult Sample with Anorexia Nervosa and Those in Recovery.

While individuals with Bulimia Nervosa (BN) and Binge Eating Disorder (BED) often present with a higher rate of Alcohol Use Disorder (AUD) than the general population, it is unclear whether this extends to AN. This cross-sectional study examined differences in alcohol-related behaviours, measured using the Alcohol Use Identification Test (AUDIT), between AN participants (n = 58), recovered AN (rec-AN) participants (n = 25), and healthy controls (n = 57). Statistical models controlled for age and ethnicity. The relationship between alcohol-related behaviours with ED psychopathology and with depression was also assessed. The findings indicated that acute AN participants were not at greater risk of AUD than healthy controls. However, rec-AN participants displayed greater total audit scores than those with acute AN, and more alcohol-related behaviours than healthy controls. Acute AN participants consumed significantly less alcohol than both the healthy control group and rec-AN group. No associations were found between ED psychopathology and alcohol-related behaviours in the AN group or rec-AN. This highlights alcohol as a potential coping mechanism following AN recovery. Clinicians should consider assessments for AUD and targeted interventions aimed at encouraging healthy coping mechanisms in this group. Future studies should look at alcohol use as a moderating factor for AN recovery.

BqsR/BqsS constitute a two-component system that senses extracellular Fe(II) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium best known as the predominant opportunistic pathogen infecting the lungs of cystic fibrosis patients. In this context, it is thought to form biofilms, within which locally reducing and acidic conditions can develop that favor the stability of ferrous iron [Fe(II)]. Because iron is a signal that stimulates biofilm formation, we performed a microarray study to determine whether P. aeruginosa strain PA14 exhibits a specific transcriptional response to extracellular Fe(II). Among the genes that were most upregulated in response to Fe(II) were those encoding the two-component system BqsR/BqsS, previously identified for its role in P. aeruginosa strain PAO1 biofilm decay (13); here, we demonstrate its role in extracellular Fe(II) sensing. bqsS and bqsR form an operon together with two small upstream genes, bqsP and bqsQ, and one downstream gene, bqsT. BqsR/BqsS sense extracellular Fe(II) at physiologically relevant concentrations (>10 µM) and elicit a specific transcriptional response, including its autoregulation. The sensor distinguishes between Fe(II), Fe(III), and other dipositive cations [Ca(II), Cu(II), Mg(II), Mn(II), Zn(II)] under aerobic or anaerobic conditions. The gene that is most upregulated by BqsR/BqsS, as measured by quantitative reverse transcription-PCR (qRT-PCR), is PA14_04180, which is predicted to encode a periplasmic oligonucleotide/oligosaccharide-binding domain (OB-fold) protein. Coincident with phenazine production during batch culture growth, Fe(II) becomes the majority of the total iron pool and bqsS is upregulated. The existence of a two-component system that senses Fe(II) indicates that extracellular Fe(II) is an important environmental signal for P. aeruginosa.

Gut commensal bacteria enhance pathogenesis of a tumorigenic murine retrovirus.

The influence of the microbiota on viral transmission and replication is well appreciated. However, its impact on retroviral pathogenesis outside of transmission/replication control remains unknown. Using murine leukemia virus (MuLV), we found that some commensal bacteria promoted the development of leukemia induced by this retrovirus. The promotion of leukemia development by commensals is due to suppression of the adaptive immune response through upregulation of several negative regulators of immunity. These negative regulators include Serpinb9b and Rnf128, which are associated with a poor prognosis of some spontaneous human cancers. Upregulation of Serpinb9b is mediated by sensing of bacteria by the NOD1/NOD2/RIPK2 pathway. This work describes a mechanism by which the microbiota enhances tumorigenesis within gut-distant organs and points at potential targets for cancer therapy.

Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.

Uncovering Factors Influencing Instructors' Decision Process when Considering Implementation of a Course-Based Research Experience.

Course-based undergraduate research experiences (CUREs) are an effective way to expose large numbers of students to authentic research, yet most laboratory courses still use traditional "cookbook" methods. While barriers to using CUREs have been captured postimplementation, little is known about the decision mindset before implementation or what features of CURE design may mitigate perceived barriers. Perception of an innovation (such as a CURE) influences the likelihood of its adoption, and diffusion of innovations theory posits that the decision to adopt is largely influenced by five perceived features of an innovation: relative advantage, compatibility, complexity, observability, and trialability. We conducted interviews with instructors considering using the Prevalence of Antibiotic Resistance in the Environment (PARE) project to assess their perceptions of CUREs and motivations for using PARE. Instructors viewed CUREs as having relative advantages over traditional methods; however, CUREs were also viewed as complex, with instructors citing multiple barriers. Instructors were motivated to use PARE because of its potential scientific impact and compatibility with their courses' structures and resources. Instructors perceived PARE to have few barriers to implementation compared with other CUREs. Designing CUREs that address common instructor barriers and drivers could increase the rate of diffusion of CUREs.

Acid and base stress and transcriptomic responses in Bacillus subtilis.

Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived <15%. Cells grown at pH 9.0 survived 40 to 100% at pH 10, whereas cells grown at pH 7.0 survived <5%. Thus, growth in a moderate acid or base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K(+)/H(+) antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.

Rapid acid treatment of Escherichia coli: transcriptomic response and recovery.

BACKGROUND: Many E. coli genes show pH-dependent expression during logarithmic growth in acid (pH 5-6) or in base (pH 8-9). The effect of rapid pH change, however, has rarely been tested. Rapid acid treatment could distinguish between genes responding to external pH, and genes responding to cytoplasmic acidification, which occurs transiently following rapid external acidification. It could reveal previously unknown acid-stress genes whose effects are transient, as well as show which acid-stress genes have a delayed response. RESULTS: Microarray hybridization was employed to observe the global gene expression of E. coli K-12 W3110 following rapid acidification of the external medium, from pH 7.6 to pH 5.5. Fluorimetric observation of pH-dependent tetR-YFP showed that rapid external acidification led to a half-unit drop in cytoplasmic pH (from pH 7.6 to pH 6.4) which began to recover within 20 s. Following acid treatment, 630 genes were up-regulated and 586 genes were down-regulated. Up-regulated genes included amino-acid decarboxylases (cadA, adiY, gadA), succinate dehydrogenase (sdhABCD), biofilm-associated genes (bdm, gatAB, and ymgABC), and the Gad, Fur and Rcs regulons. Genes with response patterns consistent with cytoplasmic acid stress were revealed by addition of benzoate, a membrane-permeant acid that permanently depresses cytoplasmic pH without affecting external pH. Several genes (yagU, ygiN, yjeI, and yneI) were up-regulated specifically by external acidification, while other genes (fimB, ygaC, yhcN, yhjX, ymgABC, yodA) presented a benzoate response consistent with cytoplasmic pH stress. Other genes (the nuo operon for NADH dehydrogenase I, and the HslUV protease) showed delayed up-regulation by acid, with expression rising by 10 min following the acid shift. CONCLUSION: Transcriptomic profiling of E. coli K-12 distinguished three different classes of change in gene expression following rapid acid treatment: up-regulation with or without recovery, and delayed response to acid. For eight genes showing acid response and recovery (fimB, ygaC, yhcN, yhjX, ymgABC, yodA), responses to the permeant acid benzoate revealed expression patterns consistent with sensing of cytoplasmic pH. The delayed acid response of nuo genes shows that NADH dehydrogenase I is probably induced as a secondary result of acid-associated metabolism, not as a direct response to cytoplasmic acidification.

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12.

BACKGROUND: In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. RESULTS: The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC). CONCLUSION: pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm.

Polyamine stress at high pH in Escherichia coli K-12.

BACKGROUND: Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. RESULTS: The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5). CONCLUSION: Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress.

Mammalian Lipopolysaccharide Receptors Incorporated into the Retroviral Envelope Augment Virus Transmission.

The orally transmitted retrovirus mouse mammary tumor virus (MMTV) requires the intestinal microbiota for persistence. Virion-associated lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10 and MMTV evasion of host immunity. However, the mechanisms by which MMTV associates with LPS remain unknown. We find that the viral envelope contains the mammalian LPS-binding factors CD14, TLR4, and MD-2, which, in conjunction with LPS-binding protein (LBP), bind LPS to the virus and augment transmission. MMTV isolated from infected mice lacking these LBPs cannot engage LPS or stimulate TLR4 and have a transmission defect. Furthermore, MMTV incorporation of a weak agonist LPS from Bacteroides, a prevalent LPS source in the gut, significantly enhances the ability of this LPS to stimulate TLR4, suggesting that MMTV intensifies these immunostimulatory properties. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission.

Osmolytes contribute to pH homeostasis of Escherichia coli.

BACKGROUND: Cytoplasmic pH homeostasis in Escherichia coli includes numerous mechanisms involving pH-dependent catabolism and ion fluxes. An important contributor is transmembrane K+ flux, but the actual basis of K+ compensation for pH stress remains unclear. Osmoprotection could mediate the pH protection afforded by K+ and other osmolytes. METHODS AND PRINCIPAL FINDINGS: The cytoplasmic pH of E. coli K-12 strains was measured by GFPmut3 fluorimetry. The wild-type strain Frag1 was exposed to rapid external acidification by HCl addition. Recovery of cytoplasmic pH was enhanced equally by supplementation with NaCl, KCl, proline, or sucrose. A triple mutant strain TK2420 defective for the Kdp, Trk and Kup K+ uptake systems requires exogenous K+ for steady-state pH homeostasis and for recovery from sudden acid shift. The K+ requirement however was partly compensated by supplementation with NaCl, choline chloride, proline, or sucrose. Thus, the K+ requirement was mediated in part by osmolarity, possibly by relieving osmotic stress which interacts with pH stress. The rapid addition of KCl to strain TK2420 suspended at external pH 5.6 caused a transient decrease in cytoplasmic pH, followed by slow recovery to an elevated steady-state pH. In the presence of 150 mM KCl, however, rapid addition of another 150 mM KCl caused a transient increase in cytoplasmic pH. These transient effects may arise from secondary K+ fluxes occurring through other transport processes in the TK2420 strain. CONCLUSIONS: Diverse osmolytes including NaCl, KCl, proline, or sucrose contribute to cytoplasmic pH homeostasis in E. coli, and increase the recovery from rapid acid shift. Osmolytes other than K+ restore partial pH homeostasis in a strain deleted for K+ transport.

pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry.

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.