Bacillus Calmette-Guérin Skin Reaction Predicts Enhanced Mycobacteria-Specific T-Cell Responses in Infants: A Post Hoc Analysis of a Randomized Controlled Trial.

Rationale: Scar formation following bacillus Calmette-Guérin (BCG) vaccination has been associated with lower all-cause mortality; the relation between scar and mycobacteria-specific protection against tuberculosis is debated. Objectives: To evaluate the association between BCG skin reaction and mycobacteria-specific immune responses. Methods: A post hoc analysis was done among 214 infants in Australia randomized to vaccination with one of three BCG vaccine strains (BCG-Denmark, BCG-Japan, or BCG-Russia) given at birth or BCG-Denmark given at 2 months of age. Measurements and Main Results: BCG skin reaction size and characteristics 10 weeks after vaccination were related to the in vitro mycobacteria-specific immune responses measured in stimulated whole blood. The size and characteristics of the skin reaction correlated positively with in vitro immune responses, even after adjusting for BCG vaccine strain and age at vaccination. Specifically, the reaction size and characteristics correlated with the proportion of mycobacteria-specific polyfunctional CD4+ T cells after stimulation with BCG and PPD and, to a lesser extent, after stimulation with Mycobacterium tuberculosis or Mycobacterium ulcerans. A similar correlation was observed with concentrations of IFN-γ, IL-2, tumor necrosis factor, and IL-13 in the supernatant after stimulation with BCG, PPD, and M. tuberculosis and to some degree for the proportions of mycobacteria-specific polyfunctional CD8+ T cells and CD107+ cytotoxic cells. Conclusions: BCG skin reaction correlated with the magnitude of mycobacteria-specific T-cell responses. As T-cell responses play a key role in defense against mycobacteria, the relationship between BCG scar formation and protection against tuberculosis should be revisited. This may also extend to the need for BCG revaccination in scar-negative individuals.Clinical trial registered with www.australianclinicaltrials.gov.au/clinical-trial-registries (ACTRN12608000227392).

Prevention of infant eczema by neonatal Bacillus Calmette-Guérin vaccination: The MIS BAIR randomized controlled trial.

BACKGROUND: Bacille Calmette-Guérin (BCG) vaccine could play a role in counteracting the rising prevalence of atopic diseases, through its beneficial off-target effects. We aimed to determine whether neonatal BCG vaccination reduces the incidence of eczema in infants. METHODS: Randomized controlled trial with 1272 infants allocated to receive BCG-Denmark or no BCG at birth. The primary outcome was the 12-month incidence of eczema based on 3-monthly questionnaires. Eczema was also assessed at a 12-month clinic visit. ClinicalTrial.gov: NCT01906853. RESULTS: The 12-month eczema incidence was 32.2% in the BCG group compared with 36.6% in the control group (adjusted risk difference (aRD) -4.3%, 95% CI -9.9% to 1.3%, multiple imputation model). In addition, comparing infants in the BCG group with the control group, 15.7% vs. 19.2% had eczema lesions at the 12-month visit (aRD -3.5%, 95% CI -8.0% to 1.0%); 35.7% vs. 39.0% reported using topical steroids (aRD -3.3, 95% CI -9.2 to 2.7); and 7.3% vs. 10.2% had severe eczema scores (aRD -3.0%, 95% CI -8.8% to 2.7%). In 344 high-risk infants (two atopic parents), the 12-month eczema incidence was 35.3% in the BCG group compared with 46.8% in the control group (aRD -11.5%, 95% CI -21.9% to -1.2%; number needed to treat 8.7, 95% CI 4.6 to 83.3). CONCLUSION: There is insufficient evidence to recommend neonatal BCG vaccination in all infants for the prevention of eczema in the first year of life; however, a modest beneficial effect was observed among high-risk infants. A single dose of BCG-Denmark soon after birth could reduce the incidence of eczema in infants with two atopic parents.

Neonatal Bacille Calmette-Guérin Vaccination and Infections in the First Year of Life: The MIS BAIR Randomized Controlled Trial.

BACKGROUND: Bacille Calmette-Guérin (BCG) vaccination has beneficial off-target effects that may include protecting against non-mycobacterial infectious diseases. We aimed to determine whether neonatal BCG vaccination reduces lower respiratory tract infections (LRTI) in infants in the Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR) trial. METHODS: In this investigator-blinded trial, neonates in Australia were randomized to receive BCG-Denmark vaccination or no BCG at birth. Episodes of LRTI were determined by symptoms reported in parent-completed, 3-month questionnaires over the first year of life. Data were analyzed by intention-to-treat using binary regression. RESULTS: A total of 1272 neonates were randomized to the BCG vaccination (n = 637) or control (n = 635) group. The proportion of participants with an episode of LRTI in the first year of life among BCG-vaccinated infants was 54.8% compared to 58.0% in the control group, resulting in a risk difference of -3.2 (95% confidence interval, -9.0 to 2.6) after multiple imputation. There was no interaction observed between the primary outcome and sex, maternal BCG, or the other prespecified effect modifiers. CONCLUSIONS: Based on the findings of this trial, there is insufficient evidence to support the use of neonatal BCG vaccination to prevent LRTI in the first year of life in high-income settings.

Neonatal BCG Vaccination Reduces Interferon-γ Responsiveness to Heterologous Pathogens in Infants From a Randomized Controlled Trial.

BACKGROUND: BCG vaccination has beneficial nonspecific (heterologous) effects that protect against nonmycobacterial infections. We have previously reported that BCG vaccination at birth alters in vitro cytokine responses to heterologous stimulants in the neonatal period. This study investigated heterologous responses in 167 infants in the same trial 7 months after randomization. METHODS: A whole-blood assay was used to interrogate in vitro cytokine responses to heterologous stimulants (killed pathogens) and Toll-like receptor (TLR) ligands. RESULTS: Compared to BCG-naive infants, BCG-vaccinated infants had increased production of interferon gamma (IFN-γ) and monokine induced by gamma interferon (MIG) (CXCL9) in response to mycobacterial stimulation and decreased production of IFN-γ in response to heterologous stimulation and TLR ligands. Reduced IFN-γ responses were attributable to a decrease in the proportion of infants who mounted a detectable IFN-γ response. BCG-vaccinated infants also had increased production of MIG (CXCL9) and interleukin-8 (IL-8), and decreased production of IL-10, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß, the pattern of which varied by stimulant. IL-1Ra responses following TLR1/2 (Pam3CYSK4) stimulation were increased in BCG-vaccinated infants. Both sex and maternal BCG vaccination status influenced the effect of neonatal BCG vaccination. CONCLUSIONS: BCG vaccination leads to changes in IFN-γ responsiveness to heterologous stimulation. BCG-induced changes in other cytokine responses to heterologous stimulation vary by pathogen.

Tenacious Endemic Typhoid Fever in Samoa.

BACKGROUND: Typhoid fever has been endemic on the island nation of Samoa (2016 population, 195 979) since the 1960s and has persisted through 2019, despite economic development and improvements in water supply and sanitation. METHODS: Salmonella enterica serovar Typhi isolates from the 2 hospitals with blood culture capability and matched patient demographic and clinical data from January 2008 through December 2019 were analyzed. Denominators to calculate incidence by island, region, and district came from 2011 and 2016 censuses and from 2017-2019 projections from Samoa's Bureau of Statistics. Data were analyzed to describe typhoid case burden and incidence from 2008 to 2019 by time, place, and person. RESULTS: In sum, 53-193 blood culture-confirmed typhoid cases occurred annually from 2008 to 2019, without apparent seasonality. Typhoid incidence was low among children age < 48 months (17.6-27.8/105), rose progressively in ages 5-9 years (54.0/105), 10-19 years (60.7-63.4/105), and 20-34 years (61.0-79.3/105), and then tapered off; 93.6% of cases occurred among Samoans < 50 years of age. Most typhoid cases and the highest incidence occurred in Northwest Upolu, but Apia Urban Area (served by treated water supplies) also exhibited moderate incidence. The proportion of cases from short-cycle versus long-cycle transmission is unknown. Samoan S. Typhi are pansusceptible to traditional first-line antibiotics. Nevertheless, enhanced surveillance in 2019 detected 4 (2.9%) deaths among 140 cases. CONCLUSIONS: Typhoid has been endemic in Samoa in the period 2008-2019. Interventions, including mass vaccination with a Vi-conjugate vaccine coadministered with measles vaccine are planned.

Biological sex influences antibody responses to routine vaccinations in the first year of life.

AIM: We investigated the effect of early-life factors, namely sex, delivery mode, feeding method and antibiotic exposure, on antibody responses to routine vaccinations administered during the first year of life. METHODS: One and seven months after the primary course of routine vaccines and 1 month after routine vaccines at 12 months of age, antibodies against 26 vaccine antigens were measured in 398 healthy infants. The geometric mean concentration (GMC) of antibodies (adjusted for effect modifiers with multiple linear regression) and the seroprotection rate for each vaccine were compared for each early-life factor. RESULTS: Sex had an influence on GMCs. Antibody concentrations were significantly lower at 7 months of age in females for tetanus and filamentous haemagglutinin and at 13 months of age for pertactin. In contrast, at 13 months of age, antibody concentrations were significantly higher in females for polio type 3, pneumococcal serotype 6A and measles. Sex did not have an influence on seroprotection rates. Delivery mode, feeding method and antibiotic exposure did not exert a substantial influence on vaccine antibody concentrations. CONCLUSION: There is a difference between males and females in the humoral response to routine vaccinations in the first year of life.

Neonatal BCG Vaccination Influences Cytokine Responses to Toll-like Receptor Ligands and Heterologous Antigens.

Background: BCG vaccination is associated with a reduction in all-cause infant mortality in high-mortality settings. The underlying mechanisms remain uncertain, but long-term modulation of the innate immune response (trained immunity) may be involved. Methods: Whole-blood specimens, collected 7 days after randomization from 212 neonates enrolled in a randomized trial of neonatal BCG vaccination, were stimulated with killed pathogens and Toll-like receptor (TLR) ligands to interrogate cytokine responses. Results: BCG-vaccinated infants had increased production of interleukin 6 (IL-6) in unstimulated samples and decreased production of interleukin 1 receptor antagonist, IL-6, and IL-10 and the chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1ß, and monocyte chemoattractant protein 1 (MCP-1) following stimulation with peptidoglycan (TLR2) and R848 (TLR7/8). BCG-vaccinated infants also had decreased MCP-1 responses following stimulation with heterologous pathogens. Sex and maternal BCG vaccination status interacted with neonatal BCG vaccination. Conclusions: Neonatal BCG vaccination influences cytokine responses to TLR ligands and heterologous pathogens. This effect is characterized by decreased antiinflammatory cytokine and chemokine responses in the context of higher levels of IL-6 in unstimulated samples. This supports the hypothesis that BCG vaccination modulates the innate immune system. Further research is warranted to determine whether there is an association between these findings and the beneficial nonspecific (heterologous) effects of BCG vaccine on all-cause mortality.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes.

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Investigation of Streptococcus salivarius-mediated inhibition of pneumococcal adherence to pharyngeal epithelial cells.

BACKGROUND: Pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. The probiotic bacterium, Streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. We investigated the mechanism(s) of inhibition using a human pharyngeal epithelial cell line (Detroit 562) following pre-administration of two different strains of S. salivarius. RESULTS: Whilst the bacteriocin-encoding megaplasmids of S. salivarius strains K12 and M18 were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. Experiments testing S. salivarius K12 and two pneumococcal isolates (serotypes 19F and 6A) showed that inhibition of 19F may involve S. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of K12 and 19F, and no inhibition occurred when K12 was prevented from contacting epithelial cells. K12-mediated inhibition of adherence by 6A may involve additional mechanisms, since no correlation was observed between adherence of K12 and 6A, and K12 could inhibit 6A adherence in the absence of cell contact. CONCLUSIONS: These results suggest that S. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. These findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.

Early gut colonization by Bifidobacterium breve and B. catenulatum differentially modulates eczema risk in children at high risk of developing allergic disease.

BACKGROUND: An altered compositional signature and reduced diversity of early gut microbiota are linked to development of allergic disease. We investigated the relationship between dominant Bifidobacterium species during the early post-natal period and subsequent development of allergic disease in the first year of life. METHODS: Faecal samples were collected at age 1 week, 1 month and 3 months from 117 infants at high risk of allergic disease. Bifidobacterium species were analysed by quantitative PCR and terminal restriction fragment length polymorphism. Infants were examined at 3, 6 and 12 months, and skin prick test was performed at 12 months. Eczema was diagnosed according to the UK Working Party criteria. RESULTS: The presence of B. catenulatum at 3 months was associated with a higher risk of developing eczema (ORadj = 4.5; 95% CI: 1.56-13.05, padj = 0.005). Infants colonized with B. breve at 1 week (ORadj = 0.29; 95% CI: 0.09-0.95, padj = 0.04) and 3 months (ORadj = 0.15; 95% CI: 0.05-0.44, padj = 0.00001) had a reduced risk of developing eczema. Furthermore, the presence of B. breve at 3 months was associated with a lower risk of atopic sensitization at 12 months (ORadj = 0.38; 95% CI: 0.15-0.98, padj = 0.05). B. breve colonization patterns were influenced by maternal allergic status, household pets and number of siblings. CONCLUSIONS: Temporal variations in Bifidobacterium colonization patterns early in life are associated with later development of eczema and/or atopic sensitization in infants at high risk of allergic disease. Modulation of the early microbiota may provide a means to prevent eczema in high-risk infants.

Mycobacteria-Specific Cytokine Responses Detect Tuberculosis Infection and Distinguish Latent from Active Tuberculosis.

RATIONALE: Current immunodiagnostic tests for tuberculosis (TB), including the tuberculin skin test and IFN-γ release assay (IGRA), have significant limitations, which include their inability to distinguish between latent TB infection (LTBI) and active TB, a distinction critical for clinical management. OBJECTIVES: To identify mycobacteria-specific cytokine biomarkers that characterize TB infection, determine their diagnostic performance characteristics, and establish whether these biomarkers can distinguish between LTBI and active TB. METHODS: A total of 149 children investigated for TB infection were recruited; all participants underwent a tuberculin skin test and QuantiFERON-TB Gold assay. In parallel, whole-blood assays using early secretory antigenic target-6, culture filtrate protein-10, and PPD as stimulatory antigens were undertaken, and cytokine responses were determined by xMAP multiplex assays. MEASUREMENTS AND MAIN RESULTS: IFN-γ, interferon-inducible protein-10 (IP-10), tumor necrosis factor (TNF)-α, IL-1ra, IL-2, IL-13, and MIP-1ß (macrophage inflammatory protein-1ß) responses were significantly higher in LTBI and active TB cases than in TB-uninfected individuals, irrespective of the stimulant. Receiver operating characteristic analyses showed that IP-10, TNF-α, and IL-2 responses achieved high sensitivity and specificity for the distinction between TB-uninfected and TB-infected individuals. TNF-α, IL-1ra, and IL-10 responses had the greatest ability to distinguish between LTBI and active TB cases; the combinations of TNF-α/IL-1ra and TNF-α/IL-10 achieved correct classification of 95.5% and 100% of cases, respectively. CONCLUSIONS: We identified several mycobacteria-specific cytokine biomarkers with the potential to be exploited for immunodiagnosis. Incorporation of these biomarkers into future immunodiagnostic assays for TB could result in substantial gains in sensitivity and allow the distinction between LTBI and active TB based on a blood test alone.

Positive autoregulation of mrkHI by the cyclic di-GMP-dependent MrkH protein in the biofilm regulatory circuit of Klebsiella pneumoniae.

UNLABELLED: Klebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence ("MrkH box"; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the -35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation. IMPORTANCE: Bacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen, Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and the mrkHI operon. In this positive-feedback loop, whereby MrkH activates its own production, K. pneumoniae has evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.

Control of acid resistance pathways of enterohemorrhagic Escherichia coli strain EDL933 by PsrB, a prophage-encoded AraC-like regulator.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes bloody diarrhea and hemolytic-uremic syndrome (HUS) and is the most prevalent E. coli serotype associated with food-borne illness worldwide. This pathogen is transmitted via the fecal-oral route and has a low infectious dose that has been estimated to be between 10 and 100 cells. We and others have previously identified three prophage-encoded AraC-like transcriptional regulators, PatE, PsrA, and PsrB in the EHEC O157:H7 EDL933 strain. Our analysis showed that PatE plays an important role in facilitating survival of EHEC under a number of acidic conditions, but the contribution of PsrA and PsrB to acid resistance (AR) was unknown. Here, we investigated the involvement of PsrA and PsrB in the survival of E. coli O157:H7 in acid. Our results showed that PsrB, but not PsrA, enhanced the survival of strain EDL933 under various acidic conditions. Transcriptional analysis using promoter-lacZ reporters and electrophoretic mobility shift assays demonstrated that PsrB activates transcription of the hdeA operon, which encodes a major acid stress chaperone, by interacting with its promoter region. Furthermore, using a mouse model, we showed that expression of PsrB significantly enhanced the ability of strain EDL933 to overcome the acidic barrier of the mouse stomach. Taken together, our results indicate that EDL933 acquired enhanced acid tolerance via horizontally acquired regulatory genes encoding transcriptional regulators that activate its AR machinery.

Evolutionary adaptation of an AraC-like regulatory protein in Citrobacter rodentium and Escherichia species.

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general.

Progressive ventilation inhomogeneity in infants with cystic fibrosis after pulmonary infection.

Measures of ventilation distribution are promising for monitoring early lung disease in cystic fibrosis (CF). This study describes the cross-sectional and longitudinal impacts of pulmonary inflammation and infection on ventilation homogeneity in infants with CF.Infants diagnosed with CF underwent multiple breath washout (MBW) testing and bronchoalveolar lavage at three time points during the first 2 years of life.Measures were obtained for 108 infants on 156 occasions. Infants with a significant pulmonary infection at the time of MBW showed increases in lung clearance index (LCI) of 0.400 units (95% CI 0.150-0.648; p=0.002). The impact was long lasting, with previous pulmonary infection leading to increased ventilation inhomogeneity over time compared to those who remained free of infection (p<0.05). Infection with Haemophilus influenzae was particularly detrimental to the longitudinal lung function in young children with CF where LCI was increased by 1.069 units for each year of life (95% CI 0.484-1.612; p<0.001).Pulmonary infection during the first year of life is detrimental to later lung function. Therefore, strategies aimed at prevention, surveillance and eradication of pulmonary pathogens are paramount to preserve lung function in infants with CF.

Assembly of the type II secretion system such as found in Vibrio cholerae depends on the novel Pilotin AspS.

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.

Early respiratory infection is associated with reduced spirometry in children with cystic fibrosis.

RATIONALE: Pulmonary inflammation, infection, and structural lung disease occur early in life in children with cystic fibrosis. OBJECTIVES: We hypothesized that the presence of these markers of cystic fibrosis lung disease in the first 2 years of life would be associated with reduced lung function in childhood. METHODS: Lung function (forced expiratory volume in the first three-quarters of a second [FEV0.75], FVC) was assessed in individuals with cystic fibrosis diagnosed after newborn screening and healthy subjects during infancy (0-2 yr) and again at early school age (4-8 yr). Individuals with cystic fibrosis underwent annual bronchoalveolar lavage fluid examination, and chest computed tomography. We examined which clinical outcomes (pulmonary inflammation, infection, structural lung disease, respiratory hospitalizations, antibiotic prophylaxis) measured in the first 2 years of life were associated with reduced lung function in infants and young children with cystic fibrosis, using a mixed effects model. MEASUREMENTS AND MAIN RESULTS: Children with cystic fibrosis (n = 56) had 8.3% (95% confidence interval [CI], -15.9 to -6.6; P = 0.04) lower FEV0.75 compared with healthy subjects (n = 18). Detection of proinflammatory bacterial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Aspergillus species, Streptococcus pneumoniae) in bronchoalveolar lavage fluid was associated with clinically significant reductions in FEV0.75 (ranging between 11.3 and 15.6%). CONCLUSIONS: The onset of lung disease in infancy, specifically the occurrence of lower respiratory tract infection, is associated with low lung function in young children with cystic fibrosis. Deficits in lung function measured in infancy persist into childhood, emphasizing the need for targeted therapeutic interventions in infancy to maximize functional outcomes later in life.

Disarming bacterial virulence through chemical inhibition of the DNA binding domain of an AraC-like transcriptional activator protein.

The misuse of antibiotics during past decades has led to pervasive antibiotic resistance in bacteria. Hence, there is an urgent need for the development of new and alternative approaches to combat bacterial infections. In most bacterial pathogens the expression of virulence is tightly regulated at the transcriptional level. Therefore, targeting pathogens with drugs that interfere with virulence gene expression offers an effective alternative to conventional antimicrobial chemotherapy. Many Gram-negative intestinal pathogens produce AraC-like proteins that control the expression of genes required for infection. In this study we investigated the prototypical AraC-like virulence regulator, RegA, from the mouse attaching and effacing pathogen, Citrobacter rodentium, as a potential drug target. By screening a small molecule chemical library and chemical optimization, we identified two compounds that specifically inhibited the ability of RegA to activate its target promoters and thus reduced expression of a number of proteins required for virulence. Biophysical, biochemical, genetic, and computational analyses indicated that the more potent of these two compounds, which we named regacin, disrupts the DNA binding capacity of RegA by interacting with amino acid residues within a conserved region of the DNA binding domain. Oral administration of regacin to mice, commencing 15 min before or 12 h after oral inoculation with C. rodentium, caused highly significant attenuation of intestinal colonization by the mouse pathogen comparable to that of an isogenic regA-deletion mutant. These findings demonstrate that chemical inhibition of the DNA binding domains of transcriptional regulators is a viable strategy for the development of antimicrobial agents that target bacterial pathogens.

Role of class 1 serine protease autotransporter in the pathogenesis of Citrobacter rodentium colitis.

A growing family of virulence factors called serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by Shigella, Salmonella, and Escherichia coli pathotypes. SPATEs are subdivided into class 1 and class 2 based on structural features and phylogenetics. Class 1 SPATEs induce cytopathic effects in numerous epithelial cell lines, and several have been shown to cleave the cytoskeletal protein spectrin in vitro. However, to date the in vivo role of class 1 SPATEs in enteric pathogenesis is unknown. Citrobacter rodentium, a natural mouse pathogen, has recently been shown to harbor class 1 and class 2 SPATEs. To better understand the contribution of class 1 SPATEs in enteric infection, we constructed a class 1 SPATE null mutant (Δcrc1) in C. rodentium. Upon infection of C57BL/6 mice, the Δcrc1 mutant exhibited a hypervirulent, hyperinflammatory phenotype compared with its parent, accompanied by greater weight loss and a trend toward increased mortality in young mice; the effect was reversed when the crc1 gene was restored. Using flow cytometry, we observed increased infiltration of T cells, B cells, and neutrophils into the lamina propria of the distal colon in mice fed the Δcrc1 mutant, starting as early as 5 days after infection. No significant difference in epithelial cytotoxicity was observed. Reverse transcription-PCR (RT-PCR) analysis of distal colonic tissue on day 10 postinfection showed significant increases in mRNA encoding cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-1ß, and inducible nitric oxide synthase (iNOS) but not in mRNA encoding IL-17, IL-4, or IL-10 in the Δcrc1 mutant-infected mice. Our data suggest a previously unsuspected role for class 1 SPATEs in enteric infection.

Susceptibility to acute rheumatic fever based on differential expression of genes involved in cytotoxicity, chemotaxis, and apoptosis.

It is unknown why only some individuals are susceptible to acute rheumatic fever (ARF). We investigated whether there are differences in the immune response, detectable by gene expression, between individuals who are susceptible to ARF and those who are not. Peripheral blood mononuclear cells (PBMCs) from 15 ARF-susceptible and 10 nonsusceptible (control) adults were stimulated with rheumatogenic (Rh+) group A streptococci (GAS) or nonrheumatogenic (Rh-) GAS. RNA from stimulated PBMCs from each subject was cohybridized with RNA from unstimulated PBMCs on oligonucleotide arrays to compare gene expression. Thirty-four genes were significantly differentially expressed between ARF-susceptible and control groups after stimulation with Rh+ GAS. A total of 982 genes were differentially expressed between Rh+ GAS- and Rh- GAS-stimulated samples from ARF-susceptible individuals. Thirteen genes were differentially expressed in the same direction (predominantly decreased) between the two study groups and between the two stimulation conditions, giving a strong indication of their involvement. Seven of these were immune response genes involved in cytotoxicity, chemotaxis, and apoptosis. There was variability in the degree of expression change between individuals. The high proportion of differentially expressed apoptotic and immune response genes supports the current model of autoimmune and cytokine dysregulation in ARF. This study also raises the possibility that a "failed" immune response, involving decreased expression of cytotoxic and apoptotic genes, contributes to the immunopathogenesis of ARF.