Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes

Abstract

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

Fig. 1

Urinary tract is an important source of A. baumannii clinical isolates. Graph depicts the distribution of isolates obtained from each anatomical site, listed by study and a “pooled total”. Studies included in the analysis are listed on the left axis, with the current study underlined^–. Percentage of pooled total isolates corresponding to each anatomical site, is noted in the top-most bar. Table along right axis lists the number of isolates included, the dates of isolate collection, and the country/region where each study was performed. *, “other” isolates comprise 7.5% of “Pooled Total” isolates. SST/MSK: soft tissue/musculoskeletal. Source data are provided as a Source Data file

Fig. 2

A. baumannii UPAB1 strain is an uropathogen. a Catheter-implanted mice were infected with ~2 × 10⁸ CFU of the indicated strains. Following 24 h of infection, total numbers of CFU recovered were determined for the implants, bladders and kidneys. Each symbol represents an individual mouse. For each strain, the median value is shown as the horizontal line. Statistical analyses were performed using the Mann–Whitney U test, *p < 0.05. b Left panel: Representative images of an infected mouse bladder with implants at 24 hpi. Sections were stained for cell nuclei (blue), fibrinogen (green), and UPAB1 (red). Dotted white lines denote the separation of bladder tissue (LP: Lamina Propria) from lumen (LM). Bar: 20 µm. Right panel: UPAB1 colocalizes with fibrinogen deposited on implants at 24 hpi. c Growth curves of UPAB1, 19606, MRSA 1369, E. coli UTI89 and E. coli W3110 in healthy pooled urine as measured by OD600. The number of independent data points represented is four. Data represent mean and standard deviation values. Source data are provided as a Source Data file

Fig. 3

UPAB1 requires CUP pili to establish CAUTI. a Genetic organization of the CUP1 and CUP2 pili locus. b Transmission electron microscopic images showing the presence of pili structures in UPAB1 grown 2 by 24 h in static conditions. The pili-like structures were mostly absent in the ∆CUP1,2 mutant strain. Bars: 500 nm. c The ∆CUP1,2 mutant strain is impaired in the establishment of CAUTI. Each symbol represents an individual mouse. Data shown are pooled from two independent experiments. For each strain, the median value is shown as the horizontal line. Statistical analyses were performed using the Mann–Whitney U test, **p < 0.005. Source data are provided as a Source Data file

Fig. 4

pAB5 is required for the establishment of CAUTI. Each symbol represents an individual mouse. Data shown are pooled from two independent experiments with five mice. For each strain, the median value is shown as the horizontal line. Statistical analyses were performed using the Mann–Whitney U test, **p < 0.005, ****p < 0.0001. Source data are provided as a Source Data file

Fig. 5

pAB5 is detrimental in an acute pneumonia murine model. Mice were intranasally inoculated with 1 × 10⁹ CFU of either the WT UPAB1 or UPAB1p- strain. Following 36 h of infection, total numbers of CFU recovered were determined for lungs, spleen, liver, kidneys and heart. Each symbol represents an individual mouse. Data shown are pooled from two independent experiments. For each strain, the mean value is shown as the horizontal line. Statistical analyses were performed using the Mann–Whitney U test, ****p < 0.0001. Source data are provided as a Source Data file

Fig. 6

pAB5 modulates chromosomally-encoded proteins. Quantitative proteome analysis of the effect of pAB5 on the secretome (a), the whole proteome with shaking (b), and whole proteome under static conditions (c). Manhattan plots demonstrating the significance of protein alteration, –log10(p-value), vs position in the genome are shown. The direction of the protein alteration is colored coded according the provided heat map. Selected proteins and accession numbers are shown. Full data sets are shown in Supplementary Fig. 7. Pink shadow highlights the genes encoding for CUP1 pili, yellow shadow highlights the genes encoding for CUP2 pili, orange shadow highlights the genes of PNAG biosynthetic pathway and green shadow highlights the genes encoding for the T6SS. Four biological replicates of each condition were analyzed. The default Maxquant FDR setting of 1% FDR at the protein and peptide levels were used

Fig. 7

pAB5 differentially modifies the expression of chromosomal genes. DEGs with adjusted p-value < 0.1 for shaking (a) and static (b) grown conditions are depicted as a volcano plot to show the relationship between Log2 fold change and statistical significance. Each circle depicts one gene, upregulated genes are blue and downregulated genes are red, with the accession number shown for select genes. Pink shadow highlights the genes encoding for CUP1 pili, yellow shadow highlights the genes encoding for CUP2 pili, orange shadow highlights the genes of PNAG biosynthetic pathway and green shadow highlights the genes encoding for the T6SS. Three biological replicates of each condition were analyzed. All genes in the transcriptomic volcano plot with adjusted p-values above the statistical cut off of 0.1 (i.e., not significantly different between conditions) per the DESeq2 vignette are in gray, while the genes with adjusted p-values below 0.1 are blue or red

Fig. 8

Effects of different LCPs in UPAB1. a Congo red agar plates, showing differences in the colony color (red versus white) associated with production of PNAG. b Western blot assays probing for Hcp (green) expression and secretion in whole-cell (W) or supernatants (S). RNA polymerase (RNAP, red) was used as a loading and lysis control. Source data are provided as a Source Data file