Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells

Abstract

Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii.

Keywords: acinetobacter baumannii; attachment; biofilm; pathogenicity; pili; virulence.

Figure 1.

Quantification of biofilm formation in 25 A. baumannii clinical isolates selected from a collection of 172 hospital-acquired strains during the 2nd Spanish Study of colonization/infection caused by A. baumannii (GEIH/REIPI-Ab2010). Experiments were performed in triplicate and each bar represents the mean ± standard deviation (* P value < 0.0001).

Figure 2.

cDNA amplification of genes from the LH92_11070-11085 operon of A. baumannii MAR002 strain. The intergenic regions from genes LH92_11070-11075, LH92_11075-11080 and LH92_11080-11085 are shown in lanes 1, 2 and 3, respectively. Genomic DNA was used as template for positive control (lanes 6 to 8, respectively). Lanes 4 and 9 show the gyrB amplification from cDNA and DNA, respectively (positive controls). Lane 5 shows the GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific).

Figure 3.

A) Quantification of biofilm formation by crystal violet staining. Eight independent replicates were done. Student´s t-test was done, values are means and bars indicate the standard deviation. B) SEM analysis of bacterial biofilm on plastic surface at the liquid-air interface of the A. baumannii strains a) ATCC 17978, b) MAR002, c) MAR002Δ11085 and d) MAR002Δ11085 complemented. All micrographs were taken at 5,000x magnification. Bars indicate the scale marks (2 μm).

Figure 4.

Quantification of bacterial adhesion to A549 human alveolar epithelial cells by A. baumannii ATCC 17978, MAR002, MAR002Δ11085 (Δ11085) and MAR002Δ11085 complemented (Δ11085 complemented). A) Percentage of attached bacteria after 3 h of infection. B) Percentage of attached bacteria after 24 h of infection. Four independent replicates were performed. T-student test were done and bars indicate the standard deviation.

Figure 5.

SEM analysis of bacterial attachment to A549 human alveolar epithelial cells. Uninfected and healthy A549 cells covered by surfactant are shown in micrograph A as a negative control. A549 cells were infected with A. baumannii ATCC 17978 strain (B), with the MAR002 parental strain (C) and (D), with the MAR002 mutant strain lacking LH92_11085 (Δ11085) (E) or the mutant strain (Δ11085) complemented (F). All micrographs were taken at 5,000x magnification. Bars indicate the scale (2 µm).

Figure 6.

TEM images of A. baumannii strains ATCC 17978 (A), MAR002 (B), MAR002Δ11085 (C) and complemented MAR002Δ11085 (D). Images were taken at 50,000x magnification. The longer thin pili present in small amounts are pointed out by white arrows. The shorter thick pili that form a dense halo around the surface are pointed by black arrows. Bars indicate the scale (500 nm).

Figure 7.

Quantification of A549 cell death caused by A. baumannii ATCC 17978, MAR002, MAR002Δ11085 (Δ11085) and MAR002Δ11085 complemented (Δ11085 complemented). Four independent replicates were performed. T-student test were done and bars indicate the standard deviation.