A heterologous cluster of glycosyltransferase genes was identified in the three Moraxella catarrhalis LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.Moraxella catarrhalis, a gram-negative diplococcus, is considered a significant cause of acute otitis media in children and lower respiratory infections in adults with chronic obstructive pulmonary disease (COPD) (12,21,29). A number of putative virulence factors have been described for M. catarrhalis (8,11,12,17,20), including a surface-exposed lipooligosaccharide (LOS) (6,18,23,32). Structural and serological studies with M. catarrhalis have described only three different LOS serotypes (termed A, B, and C), which vary in length and content of the oligosaccharide branches (3-5, 13, 15, 28). One serological study by Vaneechoutte et al. grouped clinical isolates into serotypes A (60%), B (30%), and C (5%), with 5% of the strains unidentified (28). That has been the only study to investigate the prevalence of specific M. catarrhalis LOS serotypes in the population. The difficulties with serological determinations of M. catarrhalis LOS expression are the limited quantities of antibodies, the absolute requirement for purified sample, and the potential for cross-reactivity between serotypes A and C (13,24,25).Recently, a cluster of three glycosyltransferase (lgt) genes were identified and characterized in a strain of M. catarrhalis 7169 expressing serotype B LOS (6). Primers 406 and 408 (Table 1) designed to this region were subsequently used in PCRs with chromosomal DNA from M. catarrhalis 25238, the previously defined LOS serotype A strain, and M. catarrhalis RS10, the previously defined LOS serotype C strain (Table 2) (3, 4). PCR was performed in 50-l reaction mixtures containing PCR SuperMix (Invitrogen, Carlsbad, CA), 20 pmol/l of each primer, and 1 l chromosomal DNA prepared as previously described (26). Amplifications were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol for 25 cycles with an annealing temperature of 53.1°C and extension time of 4 min. Primers 406 and 408 (Table 1) produced an amplicon of 4.3 kb in serotype A and C strains, which was 1 kb larger than the product amplified in the serotype B strain, 7169 (data not shown).Sequence analyses (MacVector 7.2 software; Accelrys, San Diego, CA) of the entire region amplified by primer 406 and flanking primer 704 (Table 1) identified an additional open reading frame upstream of the original lgt cluster described in 7169 and in the same orientation as lgt1, as illustrated in Fig. 1A and C. A ClustalW alignment with the translated sequence of this open reading frame (Lgt4) in both strains revealed 46% identity and 60% similarity to Lgt1, an ␣(1-2) glucosyltransferase in M. catarrhalis 7169 (6). The 5Ј end of this cluster...
Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.
Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of ␣-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of ␣-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.
A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) “top-down” analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein’s persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant.
Moraxella catarrhalis express three predominant forms of lipooligosaccharide (LOS) molecules on the bacterial surface. These major glycolipids contain specific carbohydrate epitopes that distinguish each glycoform into serotype A, B, or C LOS. All three serotypes, however, share a common glucose containing inner-core structure, consisting of an alpha-glucose attached to 2-keto-3-deoxyoctulosonic acid (KDO), which is unique among Gram-negative bacteria. Many of the LOS glycosyltransferase genes (lgt) responsible for assembly of the extended M. catarrhalis LOS structure have been identified. In this report, we now describe the identification and characterization of Lgt6, a unique glycosyltransferase that is responsible for the addition of the first glucose to the inner core thus initiating the assembly of full length LOS. Isogenic mutants defective in the expression of lgt6 were constructed in all three M. catarrhalis LOS serotypes and the resulting LOS glycoforms consisted of KDO(2)-lipid A-OH as analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In addition, the expression of lgt6 in trans in a heptose-deficient Neisseria meningitidis NMB gmhX mutant resulted in the addition of a hexose to the LOS of this strain. These studies demonstrate that Lgt6 functions as an alpha-(1-5)-glucosyltransferase in M. catarrhalis adding the primary glucose to the KDO(2)-lipid A-OH in LOS biosynthesis. The function of Lgt6 is required for the completion of both the major and minor oligosaccharide chains in M. catarrhalis.
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