Positive Signature-Tagged Mutagenesis in Pseudomonas aeruginosa: Tracking Patho-Adaptive Mutations Promoting Airways Chronic Infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection

Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution

The lipopolysaccharide of Haemophilus parainfluenzae

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Neoadjuvant survivin-targeted immunotherapy maveropepimut-S (MVP-S) to increase Th1 immune response in Ki67-high hormone receptor-positive (HR+) early-stage breast cancer (ESBC).

Background: HR+ ESBC is associated with suboptimal pathologic complete response rate (pCR, ̃10%) following neoadjuvant cytotoxic chemotherapy. Neoadjuvant anti-endocrine therapy with aromatase inhibitors (AI) may serve as an effective alternative. Efficacy can be gauged using the surrogate Ki67 cell proliferation histologic marker. Patients with poor Ki67 response (defined as Ki67 \u3e 10%) following neoadjuvant AI exhibit poor prognosis and therapeutic resistance to both anti-endocrine therapy and chemotherapy. In a genomic analysis among Ki67-high HR+ tumors, we identified 8-fold upregulation of BIRC5 (survivin), a gene that regulates apoptosis and the cell cycle and that is associated with poor clinical outcome. Maveropepimut-S (MVP-S, previously named DPX-Survivac) leverages the non-aqueous, lipid-based DPX delivery platform to educate a specific and persistent T cell-based immune response to 5 HLA-restricted peptides from Survivin, a cancer-associated protein commonly upregulated in several cancers. Treatment with MVP-S and intermittent, low-dose cyclophosphamide (CPA) has shown tumor infiltration of survivin-specific T cells. Previous clinical trials have shown that MVP-S is well-tolerated, immunogenic, and could lead to clinical response in several cancer indications. Further exploration of the regimen in breast cancer could extend the application of this immunotherapy for this unmet medical need. Methods: NCT04895761 is phase I trial evaluating the safety and immunologic effects of neoadjuvant MVP-S plus letrozole (arm A, n = 6), with/without tumor-directed MR-guided radiotherapy (arm B, n = 6), or intermittent low-dose cyclophosphamide or CPA (arm C, n = 6). Postmenopausal patients with T1c+ HR+HER2- breast cancer with Ki67 \u3e 10% will receive two doses of MVP-S and 7 weeks of neoadjuvant letrozole prior to surgery (all arms), whereas arm B will be treated additionally with concurrent 10Gy x 2 tumor boost radiation to facilitate immunogenic cell death, and arm C (n = 6) will be treated additionally with intermittent low-dose CPA (50mg BID) to facilitate regulatory T cell depletion. The primary objective is safety. Biomarker objectives are to evaluate for each treatment arm: 1) systemic type I survivin-specific immune response, as measured by IFN-γ ELISPOT; 2) changes in immune environment by GeoMx digital spatial genomic profiling; 3) and changes in tumor infiltrating lymphocytes (TILs) and Ki67. These data will be used to identify the most immunogenic MVP-S combination therapy for study in phase II trial powered to assess clinical outcome (pCR). Clinical trial information: NCT04895761. © 2022 by American Society of Clinical Oncolog

Darwin's “Natural Science of Babies”

In 1877, the newly founded British journal Mind published two papers on child development. The earlier, by Hippolyte Taine, prompted the second article: an account of his own son's development by the naturalist Charles Darwin. In its turn, Darwin's paper, “A Biographical Sketch of an Infant,” influenced others. Diary studies similar to Taine's and Darwin's appeared in Mind from 1878. In addition, the medical profession started to consider normal child language acquisition as a comparison for the abnormal. Shortly before his death in 1882, Darwin continued with his theme, setting out a series of proposals for a program of research on child development with suggested methodology and interpretations. Darwin, whose interest in infants and the developing mind predated his 1877 paper by at least 40 years, sought to take the subject out of the nursery and into the scientific domain. The empirical study of the young child's developing mental faculties was a source of evidence with important implications for his general evolutionary theory. The social status of children in England was the subject of considerable discussion around the time Darwin's 1877 paper appeared. Evolutionary theory was still relatively new and fiercely debated, and an unprecedented level of interest was shown by the popular press in advance of the publication. This article considers the events surrounding the publication of Darwin's article in Mind, the notebook of observations on Darwin's children (1839-1856) that served as its basis, and the research that followed publication of “Biographical Sketch.” We discuss the impact this article, one of the first infant psychology studies in English, made on the scientific community in Britain in the latter half of the nineteenth century