Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.

Keywords: Acinetobacter; Lon protease; biofilms.

FIG 1

Transposon mutagenesis screen identifies biofilm mutants with altered colony and pellicle biofilm formation. (A) Colony biofilms of parental and lon::Tn strains on BHI Congo red plates. (B) Biofilm pellicles formed in BHI medium in a 12-well untreated polystyrene dish and imaged at 48, 72, and 96 h. Panels A and B show representative images, at 96 h, of experiments performed in at least triplicate. (C) Absorbance at 570 nm for crystal violet staining, at 96 h, of polystyrene-adhered cells from pellicle biofilms formed in BHI medium. The data are representative data for experiments performed in triplicate. Error bars represent standard deviations. An unpaired two-tailed t test was used for statistical analysis, relative to the parental strain. *, P < 0.05.

FIG 2

Lon protease negatively regulates biofilm formation in A. baumannii. (A) Colony biofilms of WT, lon::FRT, and lon::FRTc strains on BHI Congo red plates. (B) Pellicle biofilms of the same strains as in panel A in BHI medium. Panels A and B show representative images, at 96 h, of experiments performed in at least triplicate. (C) Absorbance at 570 nm for crystal violet staining, at 96 h, of polystyrene-adhered cells from pellicle biofilms formed in BHI medium. The data are representative data for experiments performed in triplicate. Error bars represent standard deviations. An unpaired two-tailed t test was used for statistical analysis, relative to the WT strain. *, P < 0.05; NS, not significant.

FIG 3

Lon-deficient mutant has increased UV sensitivity and decreased motility. (A) UV (27 J/m²) survival of WT, lon::FRT, and lon::FRTc strains. Data shown are the means for experiments performed in biological duplicate, and error bars represent standard deviations. An unpaired two-tailed t test was used for statistical analysis, relative to the WT strain. *, P < 0.05; NS, not significant. (B) WT, lon::FRT, and lon::FRTc strains were spotted on modified 0.3% YT-agarose to test motility. Representative images for experiments performed in at least triplicate are shown.

FIG 4

Lon-deficient mutant upregulates a surface antigen and has unique cell morphology. (A) Separation of cell-free lysates from the WT (lane 1), lon::FRT (lane 2), lon::FRTc (lane 3), and lon::FRT A1S_1383::Km (lane 4) strains by SDS-PAGE on a 4 to 12% polyacrylamide gel in MES buffer. The same protein concentration of each lysate was loaded in the wells, to allow direct comparison between strains. Arrows point to two strongly upregulated bands. Band 1 and band 2 were identified by mass spectrometry to be enriched with A1S_1383 and A1S_2230, respectively. (B) Real-time qPCR performed in biological duplicate to determine expression of the A1S_1383 gene transcript from the lon::FRT mutant, relative to the WT strain. Expression was standardized to 16S rRNA expression, and the value for the WT strain was set to 1. Error bars represent standard deviations. An unpaired two-tailed t test was used for statistical analysis, compared to A1S_1383 expression of the WT strain. *, P < 0.05. (C) Single-cell phase microscopy of saturated cultures. Arrows point to the phase bright center. The sizes of cells were measured using MicrobeJ (62). The mean cell areas and standard deviations (n > 1,000) are provided below the images. Representative microscopic images of the strains are shown. Scale bars represent 10 μm. (D) Percentage of dead cells, among total cells, quantified at 24 h over multiple fields of view (>8 fields), with at least 2,700 cells counted. The error bars represent the 95% confidence intervals, and a chi-square test was used for statistical analysis. *, P < 0.05; NS, not significant.

FIG 5

Lon protease influences the cellular envelope. (A) Negative capsule staining (41) was performed on WT, lon::FRT, lon::FRTc, and lon::FRT surA1::Km strains. The white space surrounding cells is indicative of capsule. Representative microscopic images of the strains are shown. The thickness of the capsule was measured with ImageJ (NIH) (n = 100), and the mean thickness and standard deviation are provided below each image. Scale bars represent 10 μm. Staining was performed in three independent experiments. (B) Real-time qPCR was performed in biological triplicate to determine expression of the K locus in the lon::FRT and WT strains. Expression was standardized to that of 16S rRNA. Relative quantification of the K locus gene transcripts is shown, with the value for the WT strain being set to 1. Error bars represent standard deviations. An unpaired two-tailed t test was used for statistical analysis, compared to expression of the parental strain. NS, not significant (P > 0.05).