The Role of Type IV Pilus Proteins, PilA and PilB, in Acinetobacter Baumannii Virulence and Pathogenesis

Date

2021

Authors

Caldwell, Bayley Lynn

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Abstract

Acinetobacter baumannii is an emerging opportunistic, multidrug resistant Gram-negative bacterium which is associated with nosocomial infection as well as infections in combat troops returning from a conflict zone. Previously, our laboratory has shown that deletion of the thioredoxin A (TrxA) gene in A. baumannii reduced bacterial growth and virulence by down-regulation of Type IV pili (T4P) gene expression. Bacterial T4P secretion system contributes to DNA uptake, protein secretion and twitching motility has been reported to play an important role in pathogenesis. TrxA, a potent disulfide bond reducer, might affect T4P secretion system during assembly by targeting T4P component proteins, PilA, PilB and PilD, which contain multiple cysteine residues for proper protein folding and function.

Based on the alteration of T4P as one of the ΔtrxA phenotypes, we hypothesize that Acinetobacter baumannii lacking T4P genes (PilA, PilB, and PilD) is attenuated in virulence and pathogenesis. We first characterized morphological changes of ΔPilA, ΔPilB and ΔPilD by transmission electron microscopy and found that similar to ΔTrxA, these T4P mutant strains exhibit significant decrease of surface pili compared to the WT AB5075. However, unlike ΔTrxA, all 3 T4P mutants showed no significant cell size reduction. Next, we assess the alternation of twitching motility and biofilm formation, two critical bacterial survival functions, in T4P mutants. We observed a decreased motility in ΔTrxA and ΔPilA, but not in ΔPilB and ΔPilD. Biofilm formation were greatly reduced in all mutant strains and decrease of bacterial metabolism in the biofilm mass were evident for ΔPilA, ΔPilD and ΔTrxA. Finally, we assess bacterial pathogenesis using an insect and a murine model of Acinetobacter infection. Results of the bacterial challenge in the larvae of Galleria mellonella revealed the increased survival in ΔPilB and ΔTrxA compared to the WT. Attenuation of ΔPilB was further confirmed in mice by intraperitoneal injection of high dose (1x107 CFU) of WT and ΔPilB. While 80% mice challenged with WT succumbed to infection, all mice survived from ΔPilB challenge. Intestinal adhesions have been known to contribute to A. baumannii's pathogenesis and our previous research has shown decreased intestinal adhesion in ΔTrxA. Among the examined T4P mutants, ΔPilA and ΔPilD, but not ΔPilB, exhibit significant reduction in bacterial attachment to intestinal mucosa. Collectively, T4P deficiency in Acinetobacter baumannii resulted in attenuation of bacterial virulence and reduced bacterial pathogenesis. Further studies on the interaction of TrxA with T4P proteins will shed light on the regulatory mechanisms of TrxA-mediated bacterial virulence.

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Keywords

Acinetobacter baumannii, PilA, PilB, Type IV Pili Secretion System

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Department

Integrative Biology