The respiratory chain of Klebsiella aerogenes in urine-like conditions: critical roles of NDH-2 and bd-terminal oxidases

Abstract

Klebsiella aerogenes is an opportunistic nosocomial bacterial pathogen that commonly causes urinary tract infections. Over the past decades, K. aerogenes strains have acquired resistance to common antibiotics that has led to the rise of multidrug-resistant and even pandrug-resistant strains. Infections produced by these strains are nearly impossible to treat, which makes K. aerogenes a global priority to develop new antibiotics and there is an urgent need to identify targets to treat infections against this pathogen. However, very little is known about the metabolism and metabolic adaptations of this bacterium in infection sites. In this work, we investigated the respiratory metabolism of K. aerogenes in conditions that resemble human urine, allowing us to identify novel targets for antibiotic development. Here we describe that, unlike other gram-negative pathogens, K. aerogenes utilizes the type-2 NADH dehydrogenase (NDH-2) as the main entry point for electrons in the respiratory chain in all growth conditions evaluated. Additionally, in urine-like media, the aerobic metabolism as a whole is upregulated, with significant increases in succinate and lactate dehydrogenase activity. Moreover, our data show that the bd-I type oxidoreductases are the main terminal oxidases of this microorganism. Our findings support an initial identification of NDH-2 and bd-I oxidase as attractive targets for the development of new drugs against K. aerogenes as they are not found in human hosts.

Keywords: Enterobacter aerogenes; Klebsiella aerogenes; NDH-2; bacteria metabolism; bd-terminal oxidase; oxidase; urine.

Figure 1

K. aerogenes growth in LB and mAUM. (A) Growth curve in LB broth. (B) Growth curve in mAUM. (C) Maximum growth. (D) Growth rate. (E) Duration of lag phase. Black bars represent data from LB and white bars indicate data from mAUM. Data are expressed as the mean ± SD, n = 3. The asterisk indicates significance mAUM vs. LB (*p < 0.05, **p = 0.001) determined by t-test analysis.

Figure 2

Respiratory chain of K. aerogenes. (A) Respiratory enzymes present in the K. aerogenes genome. (B) Main respiratory enzymes used by K. aerogenes during growth in LB and mAUM.

Figure 3

Oxygen consumption rate (OCR) of isolated K. aerogenes membranes in the presence of different substrates. Membranes isolated from cells grown in LB (A) or mAUM (B). Data are expressed as average ± S.D. Black bars show LB data and white bars show mAUM data. n ≥ 3. Asterisks indicate significance between LB and mAUM (*p < 0.05) determined by t-test analyses.

Figure 4

NADH dehydrogenases in K. aerogenes membranes. (A) Oxygen consumption rate using NADH, deamino-NADH (DA-NADH) and rotenone (Rot). (B) Participation of NADH dehydrogenases in oxygen consumption rates. (C) NADH dehydrogenase activity observed in blue native gel. Label I indicates a band of approximately 132 KDa (arrow head). BSA in its different oligomeric states is used as weight marker. Black bars correspond to data obtained from LB and white bars from mAUM. Data are expressed as average ± SD. t-test statitical analyses were performed for panels (A,B) (p < 0.05), asterisks correspond to comparison between groups: (A) *NADH vs. DA-NADH; **DA-NADH vs. Rotetone; ***NADH vs. Rotenone. (B) *NDH-1 vs. NDH-2; **NDH-2 vs. NQR; ***NDH-1 vs. NQR.

Figure 5

Sequence analysis of peptides. (A) Alignment of the K. aerogenes NDH-2 dehydrogenases and the peptides identified by proteomic analysis. (B) Alignment of subunit I of K. aerogenes bd-type terminal oxidases. Black boxes indicate the sequence of the peptides found in proteomic analysis. Alignment of amino acid sequences was performed on ClustalX2.

Figure 6

Contribution of terminal oxidases to the respiratory activity of isolated K. aerogenes membranes. Oxygen consumption rate was measured under increasing concentrations of KCN. KCN tiration of LB (A) and mAUM (B) membranes using a two-component formula (see material and methods). (C) Relative contribution of bo₃ and bd-type oxidases to the respiratory activity of LB (black bars) or mAUM (white bars). Data expressed as average ± S.D. n ≥ 3.

Figure 7

Proteomic data of main NADH-dehydrogenases, NADH-independent ubiquinone-dependent dehydrogenases and terminal oxidases. Fold change ratio of mAUM data vs. LB data is shown. Dashed line indicates no change. Data represent average ± S.D., n = 3. Asterisks denote significance (p = 0.01) as determined by t-test. Non-significant change is represented by gray bars.