Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli.

Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ, which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoyl-d-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production.IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation.

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Global antibody response to Staphylococcus aureus live-cell vaccination.

The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.

Microarray-based identification of human antibodies against Staphylococcus aureus antigens.

PURPOSE: The mortality rate of patients with Staphylococcus aureus infections is alarming and urgently demands new strategies to attenuate the course of these infections or to detect them at earlier stages. EXPERIMENTAL DESIGN: To study the adaptive immune response to S. aureus antigens in healthy human volunteers, a protein microarray containing 44 S. aureus proteins was developed using the ArrayStrip platform technology. RESULTS: Testing plasma samples from 15 S. aureus carriers and 15 noncarriers 21 immunogenic S. aureus antigens have been identified. Seven antigens were recognized by antibodies present in at least 60% of the samples, representing the core S. aureus immunome of healthy individuals. S. aureus-specific serum immunoglobulin G (IgG) levels were significantly lower in noncarriers than in carriers specifically anti-IsaA, anti-SACOL0479, and anti-SACOL0480 IgGs were found at lower frequencies and quantities. Twenty-two antigens present on the microarray were encoded by all S. aureus carrier isolates. Nevertheless, the immune system of the carriers was responsive to only eight of them and with different intensities. CONCLUSION AND CLINICAL RELEVANCE: The established protein microarray allows a broad profiling of the S. aureus-specific antibody response and can be used to identify S. aureus antigens that might serve as vaccines or diagnostic markers.

Modelling antibiotic and cytotoxic isoquinoline effects in Staphylococcus aureus, Staphylococcus epidermidis and mammalian cells.

Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.