In vivo analysis of staphylococcus aureus-infected mice reveals differential temporal and spatial expression patterns of fhuD2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. The protein is part of an investigational multi-component vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression was shown to be upregulated in murine kidneys infected with S. aureus, it is unknown whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different infection niches of S. aureus might provide different environments and iron availability resulting in different fhuD2 expression pattern within different host organs. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent iron-regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a seven-day time course, as well as quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene. Using this approach, we could show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed in the temporal expression profiles, and between infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection is instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

HexR controls glucose-responsive genes and central carbon metabolism in Neisseria meningitidis

none6noNeisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection. N. meningitidis can utilize a restricted range of carbon sources, including lactate, glucose and pyruvate, whose concentration varies in host niches. Microarray analysis of N. meningitidis grown in chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most of these genes are implicated in energy metabolism and transport as well as some implicated in virulence. In particular, genes involved in glucose catabolism were up-regulated whereas genes involved in the TCA cycle were down-regulated. Several genes encoding surface exposed proteins were up-regulated in the presence of glucose, including the MafA adhesins and the Neisseria surface protein A. Our microarray analysis led to the identification of a glucose-responsive hexR-like transcriptional regulator that controls genes of the central carbon metabolism of N. meningitidis in response to glucose. We characterized the HexR regulon and showed the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites we propose a 17-bp pseudo-palindromic HexR consensus binding motif. Furthermore, N. meningitidis strains lacking hexR expression are deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogen in vivo.openAntunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, IsabelAntunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, Isabe

In vivo gene expression profiling of Staphylococcus aureus during infection informs design of stemless leukocidins LukE and -D as detoxified vaccine candidates

A.F.H. and L.L. were funded by Marie Sklodowska-Curie Intra-European Fellowships (PIEF-GA-2012-328377 and PIEF-GA-2013-623168, respectively).Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host’s immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens.Publisher PDFPeer reviewe

Multilayer regulation of Neisseria meningitidis NHBA at physiologically relevant temperatures

This research was sponsored by GlaxoSmithKline Biologicals SA. A.F.H. was funded by Marie Sklodowska-Curie Intra-European Fellowships (PIEF-GA-2012-328377).Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30–32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.Publisher PDFPeer reviewe

Absence of protein A expression is associated with higher capsule production in staphylococcal isolates

Staphylococcus aureus is a major human pathogen, and a leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has recently been proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, about 7% of which did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5′ UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both mutation, leading likely to translation alterations, and transcriptional deregulation. Furthermore, we provide evidence that SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates than SpA alone

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively.GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.PostprintPeer reviewe

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV

A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals

The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Convalescent plasma for COVID-19 in hospitalised patients : an open-label, randomised clinical trial

Background: The effects of convalescent plasma (CP) therapy in hospitalised patients with coronavirus disease 2019 (COVID-19) remain uncertain. This study investigates the effect of CP on clinical improvement in these patients. Methods: This is an investigator-initiated, randomised, parallel arm, open-label, superiority clinical trial. Patients were randomly (1:1) assigned to two infusions of CP plus standard of care (SOC) or SOC alone. The primary outcome was the proportion of patients with clinical improvement 28 days after enrolment. Results: A total of 160 (80 in each arm) patients (66.3% critically ill, 33.7% severely ill) completed the trial. The median (interquartile range (IQR)) age was 60.5 (48–68) years; 58.1% were male and the median (IQR) time from symptom onset to randomisation was 10 (8–12) days. Neutralising antibody titres >1:80 were present in 133 (83.1%) patients at baseline. The proportion of patients with clinical improvement on day 28 was 61.3% in the CP+SOC group and 65.0% in the SOC group (difference −3.7%, 95% CI −18.8–11.3%). The results were similar in the severe and critically ill subgroups. There was no significant difference between CP+SOC and SOC groups in pre-specified secondary outcomes, including 28-day mortality, days alive and free of respiratory support and duration of invasive ventilatory support. Inflammatory and other laboratory marker values on days 3, 7 and 14 were similar between groups. Conclusions: CP+SOC did not result in a higher proportion of clinical improvement on day 28 in hospitalised patients with COVID-19 compared to SOC alone