Evolution in fast forward: a potential role for mutators in accelerating Staphylococcus aureus pathoadaptation.

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 α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-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.

Use of Moraxella catarrhalis lipooligosaccharide mutants to identify specific oligosaccharide epitopes recognized by human serum antibodies.

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.

A unique glycosyltransferase involved in the initial assembly of Moraxella catarrhalis lipooligosaccharides.

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.

Enhancing Scientific Communication Through an Undergraduate Biology and Journalism Partnership.

Scientific terminology presents an obstacle to effective communication with nonscientific audiences. To overcome this obstacle, biology majors in a general microbiology elective completed a project involving two different audiences: a scientific audience of their peers and a general, nonscientific audience. First, students presented an overview of a primary research paper and the significance of its findings to a general, nonscientific audience in an elevator-type talk. This was followed by a peer interview with a student in a journalism course, in which the biology students needed to comprehend the article to effectively communicate it to the journalism students, and the journalism students needed to ask questions about an unfamiliar, technical topic. Next, the biology students wrote a summary of their article for a scientific audience. Finally, the students presented a figure from the article to their peers in a scientific, Bio-Minute format. The biology-journalism partnership allowed biology students to develop their ability to communicate scientific information and journalism students their ability to ask appropriate questions and establish a base of knowledge from which to write.

Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

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.

Multiplex PCR assay that identifies the major lipooligosaccharide serotype expressed by Moraxella catarrhalis clinical isolates.

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.