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1. An expanded toolkit of drug resistance cassettes for Candida glabrata , Candida auris , and Candida albicans leads to new insights into the ergosterol pathway

   https://doi.org/10.1128/msphere.00311-23  

   Gregor, Justin B.; Gutierrez-Schultz, Victor A.; Hoda, Smriti; Baker, Kortany M.; Saha, Debasmita; Burghaze, Madeline G.; Vazquez, Cynthia; Burgei, Kendra E.; Briggs, Scott D. (December 2023, mSphere)

   Mitchell, Aaron P. (Ed.)

   ABSTRACT The World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies, includingCandida glabrata,Candida albicans, andCandida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,NatMXandHphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit forC. glabrata,C. auris, andC. albicansto includeKanMXandBleMXwhen coupled with anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes forCandida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion ofERG3inC. glabrata,C. auris,andC. albicansprototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that anERG5deletion inC. glabratais azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when bothERG3andERG5are deleted inC. glabrata,which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates. IMPORTANCEThe increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies inCandida glabrata,Candida auris, andCandida albicanshave been mainly limited to the use ofNatMX/SAT1andHphMX/CaHygfor genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated thatNatMX/SAT1, HphMX, KanMX,and/orBleMXdrug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway ofC. glabrata,C. auris, andC. albicans. Moreover, the utility of these tools has provided new insights intoERGgenes and their relationship to azole resistance inCandida. Overall, we have expanded the toolkit forCandidapathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis. 

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2. No to Neocosmospora : Phylogenomic and Practical Reasons for Continued Inclusion of the Fusarium solani Species Complex in the Genus Fusarium

   https://doi.org/10.1128/mSphere.00810-20  

   O’Donnell, Kerry; Al-Hatmi, Abdullah M.; Aoki, Takayuki; Brankovics, Balázs; Cano-Lira, José F.; Coleman, Jeffrey J.; de Hoog, G. Sybren; Di Pietro, Antonio; Frandsen, Rasmus J.; Geiser, David M.; et al (October 2020, mSphere)

   Mitchell, Aaron P. (Ed.)

   This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous moldFusarium.Fusariumspecies can cause localized and life-threating infections in humans. Of the 70Fusariumspecies that have been reported to cause infections, close to one-third are members of theFusarium solanispecies complex (FSSC), and they collectively account for approximately two-thirds of all reportedFusariuminfections. 

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3. Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1

   https://doi.org/10.1128/JB.02593-14  

   Schallies, Karla B.; Sadowski, Craig; Meng, Julia; Chien, Peter; Gibson, Katherine E. (June 2015, Journal of Bacteriology)

   DiRita, V. J. (Ed.)

   ABSTRACT CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacteriumSinorhizobium meliloti. Loss ofcbrAresults in increased levels of CtrA as well as its phosphorylation. While many of the knownCaulobacter crescentusregulators of CtrA phosphorylation and proteolysis are phylogenetically conserved withinS. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation inC. crescentus. In order to investigate whether CtrA proteolysis occurs inS. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss ofcbrAsignificantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1^D53A). Addition of CpdR1^D53Afully suppressescbrAmutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression inS. meliloti. Importantly, thecbrAmutant symbiosis defect is also suppressed in the presence of CpdR1^D53A. Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis. IMPORTANCEThe cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. TheSinorhizobium melilotinitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis. 

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4. Type IV Pilus-Mediated Inhibition of Acinetobacter baumannii Biofilm Formation by Phenothiazine Compounds

   https://doi.org/10.1128/spectrum.01023-23  

   Vo, Nam; Sidner, Benjamin S.; Yu, Yafan; Piepenbrink, Kurt H. (August 2023, Microbiology Spectrum)

   Atack, John M. (Ed.)

   Acinetobacterinfections are a growing burden on health care systems worldwide due to increasing antimicrobial resistance through multiple mechanisms. Biofilm formation is an established mechanism of antimicrobial resistance, and its inhibition has the potential to potentiate the use of existing drugs against pathogenicAcinetobacter. 

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5. Vibrio floridensis sp. nov., a novel species closely related to the human pathogen Vibrio vulnificus isolated from a cyanobacterial bloom

   https://doi.org/10.1099/ijsem.0.005675  

   Grant, Trudy-Ann; Jayakumar, Jane M.; López-Pérez, Mario; Almagro-Moreno, Salvador (February 2023, International Journal of Systematic and Evolutionary Microbiology)

   A Gram-stain-negative, rod-shaped bacterial strain, designatedVibrio floridensisIRLE0018 (=NRRL B-65642=NCTC 14661), was isolated from a cyanobacterial bloom along the Indian River Lagoon (IRL), a large and highly biodiverse estuary in eastern Florida (USA). The results of phylogenetic, biochemical, and phenotypic analyses indicate that this isolate is distinct from species of the genusVibriowith validly published names and is the closest relative to the emergent human pathogen,Vibrio vulnificus. Here, we present the complete genome sequence ofV. floridensisstrain IRLE0018 (4 535 135 bp). On the basis of the established average nucleotide identity (ANI) values for the determination of different species (ANI <95 %), strain IRLE0018, with an ANI of approximately 92 % compared with its closest relative,V. vulnificus, represents a novel species within the genusVibrio. To our knowledge, this represents the first time this species has been described. The results of genomic analyses ofV. floridensisIRLE0018 indicate the presence of antibiotic resistance genes and several known virulence factors, however, its pathogenicity profile (e.g. survival in serum, phagocytosis avoidance) reveals limited virulence potential of this species in contrast toV. vulnificus. 

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