Assessing the Role of Cold-Shock Protein C: a Novel Regulator of Acinetobacter baumannii Biofilm Formation and Virulence

Tomlinson, Brooke R; Denham, Grant A; Torres, Nathanial; Brzozowski, R; Allen, Jessie L; Jackson, Jessica K.; Eswara, Prahathees J.; Shaw, Lindsey N.

doi:10.1128/iai.00376-22

Cited by 7 publications

(9 citation statements)

References 95 publications

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“…To determine the mechanistic role of AmsSR in A. baumannii , we next explored its regulon using RNA-seq. Given that transcription from P amsSR is minimal under standard conditions [above and ( Tomlinson et al, 2022 )], and that CCCP induces the expression of this system, we performed these experiments using the wild-type and amsR mutant grown in the presence of this compound. Upon analysis, we identified 738 genes significantly differentially expressed between the two strains at ≥2-fold ( Figure 4A ; Supplementary Table S2 ).…”

Section: Resultsmentioning

confidence: 99%

“…RNAseq experiments were performed as described previously ( Tomlinson et al, 2022 ). Briefly, synchronous cultures of the A. baumannii wild-type and amsR mutant were prepared in biological triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative real time PCR analyses were conducted as described previously, using primers in Supplementary Table S1 ( Tomlinson et al, 2022 ). Bacterial cultures were grown, and RNA extracted as described for RNA-seq experiments.…”

Section: Methodsmentioning

confidence: 99%

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The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii

et al. 2023

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In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces PamsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii

et al. 2023

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“…Moreover, in A. baumannii , the ability to form biofilm facilitates tolerance toward external stress, desiccation and antimicrobials [ 38 ]. The C109 biofilm inhibitory activity was assessed against ATCC19606 and the clinical strains 560380 (MIC = 16 mg/L), MO5 (MIC = 32 mg/L) and HU5 (MIC = 16 mg/L).…”

Section: Discussionmentioning

confidence: 99%

Bactericidal and Anti-Biofilm Activity of the FtsZ Inhibitor C109 against Acinetobacter baumannii

et al. 2022

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In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ.

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“…Primers used for qRT-PCR are listed in Table S1 . RNA samples for RNAseq analysis were pooled at equal RNA concentrations from three biological experiments as previously described ( Tomlinson et al., 2021 ; Tomlinson et al., 2022 ) prior to library preparation at a concentration of 50 ng/µl in 20 µl. Only RNA samples with RIN>6 as determined by Tapestation analysis were utilized.…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia

Simcox

Tomlinson²,

Shaw³

et al. 2023

Front. Cell. Infect. Microbiol.

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Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab’s inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRSMab, a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRSMtb, is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosRMab regulon was predicted using bioinformatics based on DosRMtb motifs however, the role and regulon of DosRSMab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRSMab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRSMab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

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Assessing the Role of Cold-Shock Protein C: a Novel Regulator of Acinetobacter baumannii Biofilm Formation and Virulence

Cited by 7 publications

References 95 publications

The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii

The novel two-component system AmsSR governs alternative metabolic pathway usage in Acinetobacter baumannii

Bactericidal and Anti-Biofilm Activity of the FtsZ Inhibitor C109 against Acinetobacter baumannii

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia

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