Uncovering the mechanisms of Acinetobacter baumannii virulence

Abstract

Acinetobacter baumannii is a nosocomial pathogen that causes ventilator-associated as well as bloodstream infections in critically ill patients, and the spread of multidrug-resistant Acinetobacter strains is cause for concern. Much of the success of A. baumannii can be directly attributed to its plastic genome, which rapidly mutates when faced with adversity and stress. However, fundamental virulence mechanisms beyond canonical drug resistance were recently uncovered that enable A. baumannii and, to a limited extent, other medically relevant Acinetobacter species to successfully thrive in the health-care environment. In this Review, we explore the molecular features that promote environmental persistence, including desiccation resistance, biofilm formation and motility, and we discuss the most recently identified virulence factors, such as secretion systems, surface glycoconjugates and micronutrient acquisition systems that collectively enable these pathogens to successfully infect their hosts.

Figure 1:. Protein secretion and export in Acinetobacter baumannii.

Secretion systems and extracellular appendages of Acinetobacter baumannii are involved in the formation of biofilms, in virulence and bacterial competition. (A) The Csu system extrudes a type I chaperone-usher pilus that is essential for the formation and maintenance of biofilms on abiotic surfaces, which may contribute to persistence in the hospital environments. The CsuA/B pilin subunits are trafficked to the outer membrane CsuD usher by CsuC chaperones. The CsuD usher facilitates polymerization of CsuA/B monomers into a pilin fiber. Pilin polymerization is initiated by the CsuE tip adhesion and the minor pilins CsuA and CsuB, all of which are also trafficked to the CsuD usher by CsuC chaperones. Biofilm-associated proteins (Bap) and an effector protein that contains an RTX-like domain are secreted by the type I secretion system (T1SS) and are involved in the formation and stability of mature biofilms. The T1SS is composed of the outer membrane protein TolC, the periplasmic adaptor protein HlyD, and the inner membrane ATPase HlyB. The Acinetobacter trimeric autotransporter (Ata) type Vc secretion system consists of a membrane-associated transporter domain and a large, repetitive passenger domain that extrudes through the transporter domain. The adhesin promotes adherence to extracellular and basal membrane components of the host. (B) The type II secretion system (T2SS) secretes multiple effectors that were shown to be required for virulence in vivo, including the lipases LipA and LipH as well as the protease CpaA^,. Novel, dedicated chaperones are required for two of the effectors, including the CpaB chaperone, which is required for the secretion of CpaA, the most abundant type II effector. A. baumannii and A. nosocomialis also produce type IV pili, surface appendages evolutionarily related to the T2SS. In A. baumannii and A. nosocomialis these two systems share a processing protein, PilD, required to process pre-pseudopilins and pre-pilins prior to assembly into the T2SS and type IV pilus, respectively. Type IV pili are also glycosylated in A. baumannii and A. nosocomialis as a possible form of antigenic variation to evade host detection. Specifically, the pilin glycan is predicted to mask the major pilin subunit, PilA, from antibody recognition; furthermore, PilA displays remarkable sequence divergence across species and even between strains leading to reduced immune recognition in the case pilin subunits are not glycosylated. (C) Contact-dependent secretion systems are used by A. baumannii for inter- and intra-species killing to eliminate bacterial competitors. Many strains of Acinetobacter have two distinct contact-dependent inhibition (CDI) systems to kill sister cells that do not have the associated immunity protein. The type VI secretion system (T6SS)^, is used for inter-species competition and contains a novel L,D-endopeptidase, TagX, which is required for transit of the machinery through the peptidoglycan layer. A large, conjugative plasmid which also contains drug-resistance genes (not indicated) regulates the expression of T6SS in some clinical isolates. The individual components of the secretion systems are indicated in all panels.

Figure 1:. Protein secretion and export in Acinetobacter baumannii.

Secretion systems and extracellular appendages of Acinetobacter baumannii are involved in the formation of biofilms, in virulence and bacterial competition. (A) The Csu system extrudes a type I chaperone-usher pilus that is essential for the formation and maintenance of biofilms on abiotic surfaces, which may contribute to persistence in the hospital environments. The CsuA/B pilin subunits are trafficked to the outer membrane CsuD usher by CsuC chaperones. The CsuD usher facilitates polymerization of CsuA/B monomers into a pilin fiber. Pilin polymerization is initiated by the CsuE tip adhesion and the minor pilins CsuA and CsuB, all of which are also trafficked to the CsuD usher by CsuC chaperones. Biofilm-associated proteins (Bap) and an effector protein that contains an RTX-like domain are secreted by the type I secretion system (T1SS) and are involved in the formation and stability of mature biofilms. The T1SS is composed of the outer membrane protein TolC, the periplasmic adaptor protein HlyD, and the inner membrane ATPase HlyB. The Acinetobacter trimeric autotransporter (Ata) type Vc secretion system consists of a membrane-associated transporter domain and a large, repetitive passenger domain that extrudes through the transporter domain. The adhesin promotes adherence to extracellular and basal membrane components of the host. (B) The type II secretion system (T2SS) secretes multiple effectors that were shown to be required for virulence in vivo, including the lipases LipA and LipH as well as the protease CpaA^,. Novel, dedicated chaperones are required for two of the effectors, including the CpaB chaperone, which is required for the secretion of CpaA, the most abundant type II effector. A. baumannii and A. nosocomialis also produce type IV pili, surface appendages evolutionarily related to the T2SS. In A. baumannii and A. nosocomialis these two systems share a processing protein, PilD, required to process pre-pseudopilins and pre-pilins prior to assembly into the T2SS and type IV pilus, respectively. Type IV pili are also glycosylated in A. baumannii and A. nosocomialis as a possible form of antigenic variation to evade host detection. Specifically, the pilin glycan is predicted to mask the major pilin subunit, PilA, from antibody recognition; furthermore, PilA displays remarkable sequence divergence across species and even between strains leading to reduced immune recognition in the case pilin subunits are not glycosylated. (C) Contact-dependent secretion systems are used by A. baumannii for inter- and intra-species killing to eliminate bacterial competitors. Many strains of Acinetobacter have two distinct contact-dependent inhibition (CDI) systems to kill sister cells that do not have the associated immunity protein. The type VI secretion system (T6SS)^, is used for inter-species competition and contains a novel L,D-endopeptidase, TagX, which is required for transit of the machinery through the peptidoglycan layer. A large, conjugative plasmid which also contains drug-resistance genes (not indicated) regulates the expression of T6SS in some clinical isolates. The individual components of the secretion systems are indicated in all panels.

Figure 1:. Protein secretion and export in Acinetobacter baumannii.

Secretion systems and extracellular appendages of Acinetobacter baumannii are involved in the formation of biofilms, in virulence and bacterial competition. (A) The Csu system extrudes a type I chaperone-usher pilus that is essential for the formation and maintenance of biofilms on abiotic surfaces, which may contribute to persistence in the hospital environments. The CsuA/B pilin subunits are trafficked to the outer membrane CsuD usher by CsuC chaperones. The CsuD usher facilitates polymerization of CsuA/B monomers into a pilin fiber. Pilin polymerization is initiated by the CsuE tip adhesion and the minor pilins CsuA and CsuB, all of which are also trafficked to the CsuD usher by CsuC chaperones. Biofilm-associated proteins (Bap) and an effector protein that contains an RTX-like domain are secreted by the type I secretion system (T1SS) and are involved in the formation and stability of mature biofilms. The T1SS is composed of the outer membrane protein TolC, the periplasmic adaptor protein HlyD, and the inner membrane ATPase HlyB. The Acinetobacter trimeric autotransporter (Ata) type Vc secretion system consists of a membrane-associated transporter domain and a large, repetitive passenger domain that extrudes through the transporter domain. The adhesin promotes adherence to extracellular and basal membrane components of the host. (B) The type II secretion system (T2SS) secretes multiple effectors that were shown to be required for virulence in vivo, including the lipases LipA and LipH as well as the protease CpaA^,. Novel, dedicated chaperones are required for two of the effectors, including the CpaB chaperone, which is required for the secretion of CpaA, the most abundant type II effector. A. baumannii and A. nosocomialis also produce type IV pili, surface appendages evolutionarily related to the T2SS. In A. baumannii and A. nosocomialis these two systems share a processing protein, PilD, required to process pre-pseudopilins and pre-pilins prior to assembly into the T2SS and type IV pilus, respectively. Type IV pili are also glycosylated in A. baumannii and A. nosocomialis as a possible form of antigenic variation to evade host detection. Specifically, the pilin glycan is predicted to mask the major pilin subunit, PilA, from antibody recognition; furthermore, PilA displays remarkable sequence divergence across species and even between strains leading to reduced immune recognition in the case pilin subunits are not glycosylated. (C) Contact-dependent secretion systems are used by A. baumannii for inter- and intra-species killing to eliminate bacterial competitors. Many strains of Acinetobacter have two distinct contact-dependent inhibition (CDI) systems to kill sister cells that do not have the associated immunity protein. The type VI secretion system (T6SS)^, is used for inter-species competition and contains a novel L,D-endopeptidase, TagX, which is required for transit of the machinery through the peptidoglycan layer. A large, conjugative plasmid which also contains drug-resistance genes (not indicated) regulates the expression of T6SS in some clinical isolates. The individual components of the secretion systems are indicated in all panels.

Acinetobacter baumannii surface-exposed glycoconjugates.

The capsular polysaccharide, glycoproteins and hepta-acylated lipooligosaccharide (LOS) all contribute to virulence of Acinetobacter baumannii. In A. baumannii and A. nosocomialis, the capsular polysaccharide and glycoproteins are formed by glycans alone or glycans attached to proteins, respectively. Glycan synthesis is initiated at the inner membrane by dedicated glycosyltransferases that transfer sugars to a phosphorylated lipid generating a lipid-linked oligosaccharide (LLO). The LLO is then flipped to the periplasm where the glycan component can be transferred by PglL, an oligosaccharyltransferase (OTase) to proteins in the periplasm or outer membrane, or to type IV pilins by TfpO, a pilin specific OTase. The LLO can also be further processed and polymerized into a repeating polysaccharide by the Wzy polymerase prior to transport to the outer membrane for the capsule. In A. baumannii the capsular polysaccharide protects cells from complement-mediated killing. A. baumannii glycoproteins contribute to virulence by enhancing biofilm formation and maintenance, while glycosylated type IV pilins have been implied to function in immune evasion, shielding the antigenic protein from antibody recognition. Finally, the hepta-acylated LOS, consisting of a core glycan and lipid A, lacks an O-antigen but directly contributes to drug and desiccation resistance.

Colistin resistance mechanisms of A. baumannii.

In Acinetobacter baumannii, colistin resistance manifests either through modifications of the lipid anchor of LOS, termed lipid A, or by the complete loss of LOS, both of which alter binding affinity of colistin. Most commonly, colistin-resistant clinical isolates harbor mutations in the two-component regulatory system PmrA-PmrB, which is associated with the addition of phosphoethanolamine (pEtN) by PmrC, and galactosamine (GalN) to lipid A that presumably alter the binding affinity of colistin. The addition of GalN is dependent on the deacetylase activity of NaxD, which is regulated by the PmrA-PmrB system. Moreover, the dual activity of the acyltransferase, LpxM, leads to the constitutive expression of a predominately hepta-acylated form of LOS, which provides increased resistance against colistin. In extreme cases, A. baumannii will acquire mutations in the lipid A biosynthetic pathway thereby halting its production. In the absence of lipid A, A. baumannii has been shown to upregulate lipoproteins, namely A1S_1944, A1S_1945 and A1S_2739, which stabilize the outer membrane. The accumulation of the capsular polysaccharide poly-β−1,6-N-Acetylglucosamine (PNAG) has also been observed in strains lacking lipid A as a proposed mechanism of membrane stabilization.