Mechanisms of Polymyxin Resistance.

Polymyxin antibiotics are increasingly being used as last-line therapeutic options against a number of multidrug resistant bacteria. These antibiotics show strong bactericidal activity against a range of Gram-negative bacteria, but with the increased use of these antibiotics resistant strains are emerging at an alarming rate. Furthermore, some Gram-negative species, such as Neisseria meningitidis, Proteus mirabilis and Burkholderia spp., are intrinsically resistant to the action of polymyxins. Most identified polymyxin resistance mechanisms in Gram-negative bacteria involve changes to the lipopolysaccharide (LPS) structure, as polymyxins initially interact with the negatively charged lipid A component of LPS. The controlled addition of positively charged residues such as 4-amino-L-arabinose, phosphoethanolamine and/or galactosamine to LPS results in a reduced negative charge on the bacterial surface and therefore reduced interaction between the polymyxin and the LPS. Polymyxin resistant species produce LPS that intrinsically contains one or more of these additions. While the genes necessary for most of these additions are chromosomally encoded, plasmid-borne phosphoethanolamine transferases (mcr-1 to mcr-8) have recently been identified and these plasmids threaten to increase the rate of dissemination of clinically relevant colistin resistance. Uniquely, Acinetobacter baumannii can also become highly resistant to polymyxins via spontaneous mutations in the lipid A biosynthesis genes lpxA, lpxC or lpxD such that they produce no LPS or lipid A. A range of other non-LPS-dependent polymyxin resistance mechanisms has also been identified in bacteria, but these generally result in only low levels of resistance. These include increased anionic capsular polysaccharide production in Klebsiella pneumoniae, expression of efflux systems such as MtrCDE in N. meningitidis, and altered expression of outer membrane proteins in a small number of species.

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection.

Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.

Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA.

Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA(-) mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.

Coxiella burnetii: turning hostility into a home.

Coxiella burnetii, the causative agent of the human disease Q fever, is a unique intracellular bacterial pathogen. Coxiella replicates to high numbers within a pathogen-derived lysosome-like vacuole, thriving within a low pH, highly proteolytic and oxidative environment. In 2009, researchers developed means to axenically culture Coxiella paving the way for the development of tools to genetically manipulate the organism. These advances have revolutionized our capacity to examine the pathogenesis of Coxiella. In recent years, targeted and random mutant strains have been used to demonstrate that the Dot/Icm type IV secretion system is essential for intracellular replication of Coxiella. Current research is focused towards understanding the unique cohort of over 130 effector proteins that are translocated into the host cell. Mutagenesis screens have been employed to identify effectors that play important roles for the biogenesis of the Coxiella-containing vacuole and intracellular replication of Coxiella. A surprisingly high number of effector mutants demonstrate significant intracellular growth defects, and future studies on the molecular function of these effectors will provide great insight into the pathogenesis of Coxiella. Already, this expanse of new data implicates many eukaryotic processes that are targeted by the arsenal of Coxiella effectors including autophagy, apoptosis and vesicular trafficking.

Lipopolysaccharide-deficient Acinetobacter baumannii shows altered signaling through host Toll-like receptors and increased susceptibility to the host antimicrobial peptide LL-37.

Infections caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious global health problem. We have shown previously that A. baumannii can become resistant to the last-line antibiotic colistin via the loss of lipopolysaccharide (LPS), including the lipid A anchor, from the outer membrane (J. H. Moffatt, M. Harper, P. Harrison, J. D. Hale, E. Vinogradov, T. Seemann, R. Henry, B. Crane, F. St. Michael, A. D. Cox, B. Adler, R. L. Nation, J. Li, and J. D. Boyce, Antimicrob. Agents Chemother. 54:4971-4977, 2010). Here, we show how these LPS-deficient bacteria interact with components of the host innate immune system. LPS-deficient A. baumannii stimulated 2- to 4-fold lower levels of NF-κB activation and tumor necrosis factor alpha (TNF-α) secretion from immortalized murine macrophages, but it still elicited low levels of TNF-α secretion via a Toll-like receptor 2-dependent mechanism. Furthermore, we show that while LPS-deficient A. baumannii was not altered in its resistance to human serum, it showed increased susceptibility to the human antimicrobial peptide LL-37. Thus, LPS-deficient, colistin-resistant A. baumannii shows significantly altered activation of the host innate immune inflammatory response.

Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-ß-1,6-N-acetylglucosamine.

We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-ß-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

Insertion sequence ISAba11 is involved in colistin resistance and loss of lipopolysaccharide in Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii are of increasing concern, largely due to the multidrug resistance of many strains. Here we show that insertion sequence ISAba11 movement can result in inactivation of the A. baumannii lipid A biosynthesis genes lpxA and lpxC, resulting in the complete loss of lipopolysaccharide production and high-level colistin resistance.

Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment.

OBJECTIVES: electrostatic forces mediate the initial interaction between cationic colistin and Gram-negative bacterial cells. Lipopolysaccharide (LPS) loss mediates colistin resistance in some A. baumannii strains. Our aim was to determine the surface charge of colistin-susceptible and -resistant A. baumannii as a function of growth phase and in response to polymyxin treatment. METHODS: the zeta potential of A. baumannii ATCC 19606 and 10 clinical multidrug-resistant strains (MICs 0.5-2 mg/L) was assessed. Colistin-resistant derivatives (MIC >128 mg/L) of wild-type strains were selected in the presence of 10 mg/L colistin, including the LPS-deficient lpxA mutant, ATCC 19606R. To determine the contribution of LPS to surface charge, two complemented ATCC 19606R derivatives were examined, namely ATCC 19606R + lpxA (containing an intact lpxA gene) and ATCC 19606R + V (containing empty vector). Investigations were conducted as a function of growth phase and polymyxin treatment (1, 4 and 8 mg/L). RESULTS: wild-type cells exhibited a greater negative charge (-60.5  ±â€Š 2.36 to -26.2  ±â€Š 2.56 mV) thancolistin-resistant cells (-49.2  ±â€Š 3.09 to -19.1  ±  2.80 mV) at mid-log phase (ANOVA, P  <  0.05). Opposing growth-phase trends were observed for both phenotypes: wild-type cells displayed reduced negative charge and colistin-resistant cells displayed increased negative charge at stationary compared with mid-logarithmic phase. Polymyxin exposure resulted in a concentration-dependent increase in zeta potential. Examination of ATCC 19606R and complemented strains supported the importance of LPS in determining surface charge, suggesting a potential mechanism of colistin resistance. CONCLUSIONS: zeta potential differences between A. baumannii phenotypes probably reflect compositional outer-membrane variations that impact the electrostatic component of colistin activity.

Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.