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The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D
(2015)
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.
BACKGROUND:
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.
RESULTS:
Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.
CONCLUSIONS:
The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ("wide" and "narrow"), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.
The opportunistic pathogen Acinetobacter baumannii is able to grow on carnitine. The genes encoding the pathway for carnitine degradation to the intermediate malic acid are known but the transporter mediating carnitine uptake remained to be identified. The open reading frame HMPREF0010_01347 (aci01347) of Acinetobacter baumannii is annotated as a gene encoding a potential transporter of the betaine/choline/carnitine transporter (BCCT) family. To study the physiological function of Aci01347, the gene was deleted from A. baumannii ATCC 19606. The mutant was no longer able to grow on carnitine as sole carbon and energy source demonstrating the importance of this transporter for carnitine metabolism. Aci01347 was produced in Escherichia coli MKH13, a strain devoid of any compatible solute transporter, and the recombinant E. coli MKH13 strain was found to take up carnitine in an energy‐dependent fashion. Aci01347 also transported choline, a compound known to be accumulated under osmotic stress. Choline transport was osmolarity‐independent which is consistent with the absence of an extended C‐terminus found in osmo‐activated BCCT. We propose that the Aci01347 is the carnitine transporter mediating the first step in the growth of A. baumannii on carnitine.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.
Identification of unique cardiolipin and monolysocardiolipin species in Acinetobacter baumannii
(2017)
Acidic glycerophospholipids play an important role in determining the resistance of Gram-negative bacteria to stress conditions and antibiotics. Acinetobacter baumannii, an opportunistic human pathogen which is responsible for an increasing number of nosocomial infections, exhibits broad antibiotic resistances. Here lipids of A. baumannii have been analyzed by combined MALDI-TOF/MS and TLC analyses; in addition GC-MS analyses of fatty acid methyl esters released by methanolysis of membrane phospholipids have been performed. The main glycerophospholipids are phosphatidylethanolamine, phosphatidylglycerol, acyl-phosphatidylglycerol and cardiolipin together with monolysocardiolipin, a lysophospholipid only rarely detected in bacterial membranes. The major acyl chains in the phospholipids are C16:0 and C18:1, plus minor amounts of short chain fatty acids. The structures of the cardiolipin and monolysocardiolipin have been elucidated by post source decay mass spectrometry analysis. A large variety of cardiolipin and monolysocardiolipin species were found in A. baumannii. Similar lysocardiolipin levels were found in the two clinical strains A. baumannii ATCC19606T and AYE whereas in the nonpathogenic strain Acinetobacter baylyi ADP1 lysocardiolipin levels were highly reduced.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. The success of A. baumannii is based on the rise of multiple antibiotic resistances and its outstanding potential to persist in the human host and under conditions of low water activity in hospital environments. Combating low water activities involves osmoprotective measures such as uptake of compatible solutes and K+. To address the role of K+ uptake in the physiology of A. baumannii we have identified K+ transporter encoding genes in the genome of A. baumannii ATCC 19606. The corresponding genes (kup, trk, kdp) were deleted and the phenotype of the mutants was studied. The triple mutant was defective in K+ uptake which resulted in a pronounced growth defect at high osmolarities (300 mM NaCl). Additionally, mannitol and glutamate synthesis were strongly reduced in the mutant. To mimic host conditions and to study its role as an uropathogen, we performed growth studies with the K+ transporter deletion mutants in human urine. Both, the double (ΔkupΔtrk) and the triple mutant were significantly impaired in growth. This could be explained by the inability of ΔkupΔtrkΔkdp to metabolize various amino acids properly. Moreover, the reactive oxygen species resistance of the triple mutant was significantly reduced in comparison to the wild type, making it susceptible to one essential part of the innate immune response. Finally, the triple and the double mutant were strongly impaired in Galleria mellonella killing giving first insights in the importance of K+ uptake in virulence.