The Stringent Stress Response Controls Proteases and Global Regulators under Optimal Growth Conditions in Pseudomonas aeruginosa

ABSTRACT The bacterial stringent stress response, mediated by the signaling molecule guanosine tetraphosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as a potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. Transcriptome sequencing (RNA-Seq) was used to investigate the transcriptome of a ppGpp-deficient strain under nonstressful, nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In the exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold change cutoff of ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems, including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity toward red blood cells in a stringent response mutant appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model showed increased survival rates of mice infected with the aprA mutant, consistent with previous observations that stringent response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together, these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria. IMPORTANCE Microorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under nonstressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such, it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa

The bacterial stringent stress response, mediated by the signaling molecule guanosine tetra-phosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. RNA-Seq was used to investigate the transcriptome of a ppGpp-deficient strain under non-stressful nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold-change cut-off ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity towards red blood cells, in a stringent response mutant, appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model, showed increased survival rates of the aprA mutant consistent with previous observations that stringent-response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria.Significance StatementMicroorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under non-stressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent-response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

Lack of detectable neoantigen depletion in the untreated cancer genome

ABSTRACTSomatic mutations in cancer can result in the presentation of mutated peptides on the cell surface, eliciting an immune response. Mutant peptides are presented via HLA molecules and are known as neoantigens. It has been suggested that selection acts against the underlying mutations, leading to neoantigen depletion. Knowing the extent of this specific form of immunoediting may provide fundamental insights into tumour-immune interactions during tumour evolution. Here, we quantified the extent of neoantigen depletion in a wide range of human cancers by studying somatic mutations in the HLA-binding annotated exome, i.e. genomic regions that can be translated into presented peptides. We initially observed reduced non-synonymous mutation rates in presented regions, suggestive of neoantigen depletion. However, when compared to the expected mutation rates from a trinucleotide-based mutational signature model, depletion signals were negligible. This is explained by correlative relationships between the likelihood of mutagenesis in different nucleotide sequences and predicted HLA affinities for corresponding peptides. Our results suggest that signals of immunogenic negative selection are weak or absent in cancer genomics data and that other mechanisms to escape immune responses early during tumour evolution might be more efficient.