RecA levels modulate biofilm development inAcinetobacter baumannii

Abstract

9Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is a leading cause of hospital-10 acquired infections. A. baumannii is difficult to eradicate from hospitals due to its propensity to 11 quickly gain antibiotic resistances and ability to robustly survive on dry surfaces. These strategies are 12 largely mediated by mutagenesis and biofilm development, respectively. Mutagenesis is partly 13 governed by the DNA damage response (DDR). Biofilms are multicellular communities, often 14 surface-attached, that are more difficult to eradicate than free-living planktonic cells. There is 15 increasing evidence that the DDR and biofilm development are linked processes. Here, we show that 16 upon DNA damage, the relative intracellular concentration of RecA, the key DDR protein, is lower 17 than those of Escherichia coli. Notably, we report that RecA negatively influences biofilm 18 development. Cells lacking RecA (∆recA), that are unable to upregulate the DDR, have increased 19 surface attachment and sugar content within the biofilm matrix. We further show that in A. 20 baumannii, a modest increase in RecA concentrations, akin to DDR induction, decreases surface-21 attachment. Importantly, biofilms formed by ∆ recA cells are more difficult to eradicate with 22 2 antibiotic treatment. The evidence suggests that the A. baumannii DDR influences survival 23 independent from mutagenesis. It also demonstrates the importance of understanding fundamental 24 biology to better appreciate the relationships between different bacterial survival strategies. 25 Introduction 26Acinetobacter baumannii is an emerging Gram-negative opportunistic pathogen and one of the 27 ESKAPE pathogens, a group of bacteria responsible for the majority of hospital-acquired infections 28 1 . Interest in these bacteria has stemmed from A. baumannii outbreaks in hospitals worldwide that are 29 difficult to eradicate, due to increased multi-drug resistance (MDR) 2 and resistance to desiccation 3 . 30A. baumannii is very dangerous to immunocompromised individuals, causing different illnesses, 31including pneumonia, septicemia, and wound infections 4 . 32One response pathway that underlies MDR is the DNA damage response (DDR). When gene 33 products involved in antibiotic binding or processing are mutated, resistance is acquired 5 . 34Mutagenesis can result from induction of error-prone DNA polymerase genes which are part of the 35 DDR regulon, a strategy used by different bacteria 6,7 . In Escherichia coli and many other bacteria, 36 the cells' main recombinase, RecA, and the global transcriptional repressor, LexA, manage the DDR, 37 also known as the SOS response 8 . In A. baumannii, however, there is no known LexA homologue, 38 making the A. baumannii DDR circuitry unique 9,10 . We have shown that in A. baumannii expression 39 of multiple error-prone polymerases and clinically-relevant antibiotic resistance acquisition are 40 dependent on RecA 11 . 41A. baumannii is notorious for its ability to robustly survive on surfaces. Under dry conditions, A....

The metabolite vanillic acid regulates Acinetobacter baumannii surface attachment

“…We expected the double deletion strain to show little to no biofilms if the ΔvanR high biofilms were due to high Csu. Indeed, this strain lost its high biofilm phenotype and showed the same low surface adherence phenotype observed in the single Δcsu (MB1136, Ching et al, 2023) knockout (Figure 4d).…”

“…Plasmids were created using Golden Gate as described previously (Brychcy et al, 2023). Specifically, the following oligonucleotides were used: vancat_BsmBI_new_f and vancat_r for the vanABKP complement(pMB1037); VanR_f and VanR_r for the vanR complemement (pMB972 and pMB1149); pVanAB_f and pVanAB_r for the vanAB transcriptional reporter (pMB925); pVanr_r and pVanR_f for the vanR transcriptional reporter and complement (pMB892, pMB972 and pMB1149) and csu_operon_comlement_f and csu_op-eron_complement_r for the csu complement (pMB1147, the single csu complement was described before (Ching et al, 2023) and pMB1149, the double csu vanR complement created in this work) (Table S1 for oligonucleotides and Table S2 for plasmids). Standard Golden Gate cloning in combination with the AspFlex (Brychcy et al, 2023) and EcoFlex (Moore et al, 2016) plasmids was subsequently used.…”

“…There have been numerous studies on biofilm formation in A. baumannii (Azizi et al., 2016; Chen et al., 2017; Ching et al., 2023; Espinal et al., 2012; Gaddy & Actis, 2009; Luo et al., 2015; Moon et al., 2017; Pakharukova et al., 2018; Richmond et al., 2016; Tomaras et al., 2003, 2008; Yang et al., 2022). Cell attachment in most strains, like ATCC 17978 (Moon et al., 2017), ATCC 19606 (Pakharukova et al., 2018) or AB5075 (Ahmad et al., 2023), is based on the unique archaic chaperon‐usher pili comprising CsuA/B, A, B, C, D, E (ACX60_RS06480 – ACX60_RS06505) proteins encoded in a single operon.…”

Distinct Screening Approaches Uncover PA14_36820 and RecA as Negative Regulators of Biofilm Phenotypes in Pseudomonas aeruginosa PA14

“…Similar logic appears to be present in Acinetobacter baumannii , where deletion of recA increases biofilm formation via bfmR , a global biofilm regulator, through a protein called UmuDAb. While A. baumannii lacks LexA ( 44 – 46 ), UmuDab is a known repressor ( 47 , 48 ) that appears unique to this species ( 49 ). However, unlike the repressor function of LexA, Ching et al proposed that UmuDab might be an activator of bfmR ( 44 ).…”

“…While A. baumannii lacks LexA ( 44 – 46 ), UmuDab is a known repressor ( 47 , 48 ) that appears unique to this species ( 49 ). However, unlike the repressor function of LexA, Ching et al proposed that UmuDab might be an activator of bfmR ( 44 ).…”