Abam virus

FENG ret. GRANT dec. WANG A. HUNT ret. TRAN ret. FENG J. KOCH ret. PAYNE ret. BELAY ret. SHAH ret. Contact: R. STACK ret. KOCH, R. BIER ret. RUDE ret. CHU ret. CHEN, K.

WEBER ret. LIN ret. KING ret M. KING ret. LIN, P. KING M. This version is archived content. Consistent with this model, abaM expression was found to be negatively autoregulated but positively regulated by QS. Similar findings have been reported for rsaM1 and rsaM2 from Burkholderia thailandensis Reverse transcriptase-PCR see Fig. S4 in the supplemental material confirm that abaM and abaI in A.

These results are consistent with an IFFL circuit, although further work will be required to fully characterize its properties and control of genes coregulated by QS and AbaM. Phenotypic characterization of the A. Previous studies have shown that rsaM orthologues are required for full virulence in plants 24 , 26 , but this is, to the best of our knowledge, the first time that an rsaM -like gene has been reported to be required for full virulence in a human pathogen albeit in an insect infection model.

The contribution of Bc RsaM in B. However, deletion of Bc RsaM reduced both swarming and surface attachment, the opposite to that observed for the abaM mutant.

Interestingly, both biofilm formation and surface motility in Acinetobacter have been associated with increased virulence 10 , Genetic complementation of the abaM mutant was achieved for surface motility, biofilm formation, and AHL production but not for virulence in Galleria mellonella.

Similar observations have been previously reported for other abaM orthologues, most notably tofM 26 and Bc RsaM 28 , leading to the suggestion that RsaM-like proteins may be cis -acting regulators To further define the role of QS in A. The mutant did not produce any detectable AHLs, consistent with previous studies and bioinformatic analysis indicating that A.

In AB, biofilm formation in the opaque variant of the abaI ::T26 mutant was not significantly different from with the wild type. However, it increased well above the wild type in response to exogenous OHC12, consistent with other work on the Acinetobacter AHL synthase 18 , Previous studies on QS and biofilm formation in Acinetobacter have been performed with strains that were, in contrast to AB, either not phase variable or not known to be phase variable.

Since the experiments performed in this study were all carried out with the opaque Acinetobacter variant, we also investigated biofilm formation by the translucent variant. Figure S5 shows that the abaI translucent variant produced less biofilm than the wild type. However, biofilm formation increased for both opaque and translucent abaI variants in response to exogenous OHC Furthermore, our results suggest that QS does not play an important role in virulence in the G.

While this is not unprecedented 38 , the role of Acinetobacter QS in virulence is still not well defined, and other studies suggest that QS may play an important role, depending on the strain and infection model used 21 , Overall, our data suggest that for strain AB QS is important in surface motility and biofilm formation but not virulence.

However, further work is required to fully elucidate the role of QS in the pathogenesis of Acinetobacter infection and any cross talk with other regulatory networks.

Here, we performed RNA-seq on A. This operon encodes the proteins responsible for the synthesis of the Csu pilus, a type I chaperone-usher pilus involved in attachment and biofilm formation 39 , — Moreover, the abaM ::T26 mutant also showed higher expression of some genes of the acinetin biosynthetic operon, which has also been linked to biofilm formation in A.

A previous comparison of the transcriptomes of the multidrug resistant clinical A. However, apart from abaI , no other common differentially regulated genes could be identified when the A abaI and the abaI mutant transcriptomes are compared.

This may be because of the different strains and growth conditions and sampling times used. In addition, the abaI mutant reported by Ng et al. This raises the possibility of a secondary mutation contributing to the transcriptome data, which was not validated by chemical or genetic complementation with OHC12 or abaI , respectively.

In the transcriptomic experiments presented here, the abaI transcripts were found in larger amounts in the abaI mutant in contrast to the abaI :: lux promoter fusion data where, as expected, abaI expression was lower in the abaI mutant and stimulated by provision of exogenous OHC However, the regulation of A. In addition, the promoter fusion assays were carried out over the entire growth curve in well plates, whereas the RNA was prepared from cells at a single time point grown in larger volumes.

It is also possible that the transposon insertion in abaI impacts the amount and stability of the transcript. For abaM , we found that promoter activity and transcript levels mutant were similar. This was just below the cutoff applied to our data see Table S4.

In the abaI mutant, abaM was not differentially expressed at the late stationary time point chosen for the RNA-seq. Consequently, future work will be required to unravel these observations with respect to abaI and abaM regulation, particularly in the context of growth environment.

Overall, the work described here establishes that AbaM plays a central role in regulating QS, surface motility, biofilm formation, and virulence. The apparently contradictory regulatory impact of abaM and abaI mutations that result in either increased or no AHL production, respectively, on the expression of genes such as the csu cluster can be explained as follows.

In an abaM mutant csu expression is also increased since AbaM is absent Fig. Further work will be required to elucidate the biochemical function and mechanism of action of AbaM and the RsaM protein family in general. The strains and plasmids used are listed in Table S1 in the supplemental material.

OHC12 was synthesized as described previously The opaque and translucent phases of the wild type and mutants were separated as described by Tipton et al. Plasmid pMQM see Table S1 was obtained by digesting pMQ with PmlI to remove the genes required for yeast replication and religating the resulting large linear product. MiniTn 7 T-based constructs were inserted into A.

The miniTn 7 transposon 46 carrying the abaM promoter- lux operon fusion was inserted into A. Strains to be tested were inoculated into 1. Biofilms were quantified by staining with 0. Assays were performed as described previously At least 10 larvae were used for each strain and assay. Samples were sent for bp paired-end sequencing via an Illumina platform and bioinformatic analysis to NovoGene Hong Kong, China. Negative controls lacking template or RNA incubated without reverse transcriptase were included.

The housekeeping gene rpoB was used as endogenous control for normalization. The PCR products were run in a 1. We thank Nigel M. Rather from Emory University for his guidance and advice on Acinetobacter phase variation.

This study was supported via Wellcome Trust joint senior investigator awards to M. Supplemental material is available online only. National Center for Biotechnology Information , U. Journal List J Bacteriol v. J Bacteriol. Published online Mar Prepublished online Jan Alexander , b and Paul Williams a.

Morgan R. Yves V. Brun, Editor Yves V. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address correspondence to Paul Williams, ku. AbaM regulates quorum sensing, biofilm formation, and virulence in Acinetobacter baumannii. J Bacteriol e Received Nov 17; Accepted Jan 8. This is an open-access article distributed under the terms of the Creative Commons Attribution 4. This article has been cited by other articles in PMC.

Associated Data Supplementary Materials Supplemental file 1. Open in a separate window. FIG 1. AHL production is enhanced in an abaM mutant. FIG 2. Contribution of QS and abaM to surface motility, biofilm formation, and virulence.

FIG 3. Genetic complementation of the abaM mutant phenotypes. Transcriptomic analysis of abaI ::T26 and abaM ::T FIG 4. FIG 5. Regulation of abaM. FIG 6. FIG 7. Construction of a genetically complemented abaM ::T26 strain. Biofilm assays. The effects on viral load and on reduction in hospitalizations and ER visits, and on safety, were similar in patients receiving any of the three bamlanivimab doses.

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