William R. Schwan

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments

The Gram‐positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature‐tag transposon mutagenesis screening of a 1520‐member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

Differential Bacterial Survival, Replication, and Apoptosis-Inducing Ability of Salmonella Serovars within Human and Murine Macrophages

ABSTRACT Salmonella serovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections by Salmonella enterica serovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast, S. enterica serovar Typhimurium and S. entericaserovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars of Salmonellawere tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A.1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinctSalmonella serovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar of Salmonellais linked to the ability of that serovar both to persist within and to elicit damage in a specific hosts macrophage cells.

Osmolarity and pH growth conditions regulate fim gene transcription and type 1 pilus expression in uropathogenic Escherichia coli.

ABSTRACT A comparative study was performed to determine the effects of pH, osmolarity, and human urine on the transcription of several fim genes, as well as the overall expression of type 1 pili. Several fim-lacZYA fusions were constructed on single-copy plasmids to test a range of pHs and a range of osmolarities. Growth in acidic medium slightly reduced expression from all of the fim promoters (fimA, fimB, and fimE). Increased osmolarity in neutral-pH medium repressed fimA and fimB transcription by approximately 50% when 400 mM NaCl was used and nearly threefold when 800 mM NaCl was used, whereas fimE transcription rose slightly as the osmolarity increased. This effect was more pronounced in high-osmolarity acidic media; fimB and fimA expression decreased fivefold in growth media containing 800 mM NaCl compared to expression in growth media without added NaCl. Moreover, fimE expression doubled under the same high-osmolarity conditions compared to expression in a low-osmolarity acidic environment. When a fimB-lacZ or fimE-lacZ fusion was inserted into the chromosome of strain AAEC189, fimE expression changed slightly as the osmolarity increased, but fimB expression decreased by 50% in a low-pH high-osmolarity environment. When strain AAEC189 with either a plasmid-borne fimB-lacZ fusion or a plasmid-borne fimE-lacZ fusion was grown in human urine, similar changes in the levels of fimB and fimE expression were observed. Limiting-dilution reverse transcription-PCR confirmed that these changes in fim expression occurred in clinical isolates of uropathogenic Escherichia coli grown in media with different pHs and different osmolarities. Furthermore, the invertible switch region in uropathogenic strain NU149 shifted from favoring the phase-on position in a neutral-pH low-osmolarity environment to favoring the phase-off position in a low-pH high-osmolarity environment. Results obtained with an ompR mutant strain demonstrated that fimB expression was derepressed and that OmpR may neutralize repression by an acid response regulator of fimE expression in a low-pH environment. In addition, H-NS was verified to be important in regulation of fimB, but it had only a slight effect on fimE under the specific pH and osmotic growth conditions tested. Enzyme immunoassays with anti-type 1 pilus antibody and hemagglutination assays showed that fewer type 1 pili were detected with cells in a low-pH high-osmolarity environment. Together, these observations demonstrate that a combination of low pH and high osmolarity regulates the transcription of fim genes, which favors a shift in the invertible element to the phase-off orientation and a loss of type 1 pilus expression. Taken together, our data suggest that the environmental cues that we tested may regulate expression of type 1 pili in specific in vivo niches, such as murine kidneys and possibly human kidneys.

Loss of hemolysin expression in Staphylococcus aureus agr mutants correlates with selective survival during mixed infections in murine abscesses and wounds

During the screening of a Staphylococcus aureus signature-tagged mutagenesis library, it was noted that nonhemolytic bacteria became more abundant as time passed in murine abscess and wound models, but not within organ tissues associated with systemic infections. To examine this further, a mixed population of hyperhemolytic, hemolytic, and nonhemolytic S. aureus strain RN6390 cells were inoculated into mice using abscess, wound, and systemic models of infection. After 7 days in the abscess, the hyperhemolytic group markedly declined, whereas the nonhemolytic population increased significantly. A similar phenomenon occurred in murine wounds, but not during the systemic infection. Sequencing of several of the signature-tagged mutants indicated mutations in the agrC gene or within the agrA-agrC intergenic region. Both alpha-hemolysin and delta-hemolysin activity was curtailed in these mutants, but beta-hemolysin activity was unaffected. Single strain comparisons between wild-type strain 8325-4 and strain DU1090 (hla-) as well as between strain RN6911 (agr) and wild-type strain RN6390 were performed using the same three animal models of infection. The agr mutant strain and the hla mutant strain showed no difference in bacterial counts in murine wounds compared to their respective parent strains. The same held true in murine abscesses at day 4, but strain RN6911 counts then declined at day 7. Considerable clearing of the hla mutant strain and the agr mutant strain occurred in the systemic model of infection. Mixed infections with the DU1090 and 8325-4 strains in the abscess model showed a slight advantage given to the DU1090 population, but a distinct selection for the parental 8325-4 strain in the liver. These results suggest that agr mutations cause reductions in the expression of several secreted proteins, including alpha- and delta-hemolysin, which in turn contribute to a growth advantage of this agr mutant group within a mixed population of S. aureus cells residing in abscesses and wounds.

Virulence Genes and Genotypic Associations in Nasal Carriage, Community-Associated Methicillin-Susceptible and Methicillin-Resistant USA400 Staphylococcus aureus Isolates

ABSTRACT It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.

Transcriptional Activation of the Staphylococcus aureus putP Gene by Low-Proline-High Osmotic Conditions and during Infection of Murine and Human Tissues

ABSTRACT Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 μM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.

Antibacterial Compounds from Mushrooms II: Lanostane Triterpenoids and an Ergostane Steroid with Activity Against Bacillus cereus Isolated from Fomitopsis pinicola

Anti- Bacillus cereus bioassay-guided fractionation of a crude extract of the American mushroom, Fomitopsis pinicola, was performed using thin-layer chromatography, Sephadex LH-20 column chromatography, and preparative-scale HPLC. Five lanostane triterpenoids (1-5) and one ergostane steroid (6) were isolated and identified. Compound 1 is a new lanostane triterpenoid, and its structure was determined using 1D and 2D NMR experiments, HR-MS, and physical data. Each of the purified compounds (1-6) was tested for antibacterial activity against B. cereus using standard MIC assays. Compounds 1-6 had MIC values of 32, 16, 32, 32, 128, and 64 microg/mL, respectively.

New classes of Gram-positive selective antibacterials: inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis.

Abstract An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillus anthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure–activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.

Down-regulation of the kps region 1 capsular assembly operon following attachment of Escherichia coli type 1 fimbriae to D-mannose receptors.

ABSTRACT A differential-display PCR procedure identified the capsular assembly gene kpsD after Escherichia coli type 1 fimbrial binding to mannose-coated Sepharose beads. Limiting-dilution reverse-transcribed PCRs confirmed down-regulation of the kpsD gene, and Northern blot and lacZ fusion analyses showed down-regulation of the kpsFEDUCS region 1 operon. KpsD protein levels fell, and an agglutination test showed less K capsular antigen on the surface following the bacterial ligand-receptor interaction. These data show that binding of type 1 fimbriae (pili) to d-mannose receptors triggers a cross talk that leads to down-regulation of the capsule assembly region 1 operon in uropathogenic E. coli.

OmpR regulation of the uropathogenic Escherichia coli fimB gene in an acidic/high osmolality environment.

Uropathogenic Escherichia coli (UPEC) causes more than 90 % of all human urinary tract infections through type 1 piliated UPEC cells binding to bladder epithelial cells. The FimB and FimE site-specific recombinases orient the fimS element containing the fimA structural gene promoter. Regulation of fimB and fimE depends on environmental pH and osmolality. The EnvZ/OmpR two-component system affects osmoregulation in E. coli. To ascertain if OmpR directly regulated the fimB gene promoters, gel mobility shift and DNase I footprinting experiments were performed using OmpR or phosphorylated OmpR (OmpR-P) mixed with the fimB promoter regions of UPEC strain NU149. Both OmpR-P and OmpR bound weakly to one fimB promoter. Because there was weak binding to one fimB promoter, strain NU149 was grown in different pH and osmolality environments, and total RNAs were extracted from each population and converted to cDNAs. Quantitative reverse-transcriptase PCR showed no differences in ompR transcription among the different growth conditions. Conversely, Western blots showed a significant increase in OmpR protein in UPEC cells grown in a combined low pH/high osmolality environment versus a neutral pH/high osmolality environment. In a high osmolality environment, the ompR mutant expressed more fimB transcripts and Phase-ON positioning of the fimS element as well as higher type 1 pili levels than wild-type cells. Together these results suggest that OmpR may be post-transcriptionally regulated in UPEC cells growing in a low pH/high osmolality environment, which regulates fimB in UPEC.