Effect of Subinhibitory Concentrations of Antibiotics and Disinfectants on ISAba-Mediated Inactivation of Lipooligosaccharide Biosynthesis Genes in Acinetobacter baumannii

Inactivation of the lipooligosaccharide (LOS) biosynthesis genes lpxA, lpxC and lpxD by ISAba insertion elements results in high-level resistance to colistin in A. baumannii. In the present study, we quantify the rate of spontaneous insertional inactivation of LOS biosynthesis genes by ISAba elements in the ATCC 19606-type strain and two multidrug clinical isolates. Using insertional inactivation of lpxC by ISAba11 in the ATCC 19606 strain as a model, we determine the effect of several subinhibitory concentrations of the antibiotics, namely tetracycline, ciprofloxacin, meropenem, kanamycin and rifampicin, as well as the disinfectants ethanol and chlorhexidine on ISAba11 insertion frequencies. Notably, subinhibitory concentrations of tetracycline significantly increased ISAba11 insertion, and rifampicin completely inhibited the emergence of colistin resistance due to ISAba11 inactivation of lpxC. Sequencing of ISAba11 insertion sites within the lpxC gene demonstrated that insertions clustered between nucleotides 382 and 618 (58.3% of unique insertions detected), indicating that this may be a hotspot for ISAba11 insertion. The alignment of insertion sites revealed a semi-conserved AT-rich consensus sequence upstream of the ISAba11 insertion site, suggesting that ISAba11 insertion sites may be sequence-dependent. This study explores previously uncharacterized aspects regarding the acquisition of colistin resistance through insertional activation in LOS biosynthesis genes in A. baumannii.This research was supported by grants MPY 380/18 from the Instituto de Salud Carlos III (ISCIII) awarded to M.J.M. A.C.L. is supported by the Atracción de Talento Program of the Community of Madrid.S

Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state.

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.We thank the colleagues at the CNIO Melanoma Group, as well as those at the laboratories of H. Peinado and Manuel V. (CNIO), for help and support, I. Blanco, S. Ruiz, V. Granda, S. Rueda (CNIO) and the Animal Facility, Histopathological Unit, Confocal Microscopy Unit and Crystallography and Protein Engineering Unit of CNIO for assistance with the mouse colonies and histopathological and protein analyses, and D. Sancho (CNIC) for the B16-OVAGFP cells and OT-I mouse strain, and for scientific guidance. P. Turko (University of Zurich) provided advice on the statistical analyses of tissue microarrays. We also thank the donors and the Biobank Hospital Universitario Puerta De Hierro Majadahonda (HUPHM)/Instituto De Investigacion Sanitaria Puerta De Hierro-Segovia De Arana (IDIPHISA) (PT17/0015/0020 in the Spanish National Biobanks Network) for the human specimens used in this study. M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2017-89533-R), Team Science and Established Investigator awards by the Melanoma Research Alliance, and grants from Worldwide Cancer Research and Fundacion 'La Caixa' Health Research 2019. M.S.S., P.O.-R. and J.L.R.-P. are funded by a collaborative grant from the Asociacion Espanola Contra el Cancer (AECC). D.O. is funded by grants from the Spanish Ministry of Health (AES-PIS PI18/1057) and 'Fundacion BBVA-Becas Leonardo a Investigadores y Creadores Culturales 2018'. D.C.-W. was a recipient of a predoctoral fellowship from Fundacion 'La Caixa' and is currently funded by the AECC. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. M.C.-A. and X.C. were funded by the Immutrain Marie Skodowska-Curie ITN Grant. S.H. received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement numberS