• Abstract and Figures
• Public Full-text

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2 Izabela Sitkiewicz*, Michal J. Nagiec*†, Paul Sumby*, Stephanie D. Butler‡, Colette Cywes-Bentley§, and James M. Musser*¶ *Center for Molecular and Translational Human Infectious Diseases Research, Methodist Hospital Research Institute, Houston, TX 77030; ‡Southwest Foundation for Biomedical Research, San Antonio, TX 78227; and §Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Richard M. Krause, National Institutes of Health, Bethesda, MD, September 1, 2006 (received for review July 20, 2006) The molecular basis of pathogen clone emergence is relatively frequency and severity of serotype M3 invasive infections (3). poorly understood. Acquisition of a bacteriophage encoding a Humans with GAS infections seroconvert to SlaA, indicating previously unknown secreted phospholipase A2 (designated SlaA) that this protein is made during infection (3). In addition, SlaA has been implicated in the rapid emergence in the mid-1980s of a has enzymatic activity against several phospholipid head groups new hypervirulent clone of serotype M3 group A Streptococcus. and acyl chains located at the sn-2 position (14). For example, Although several lines of circumstantial evidence suggest that SlaA SlaA cleaves and releases arachidonic acid, a potent mediator of is a virulence factor, this issue has not been addressed experimen- the inflammatory cascade. tally. We found that an isogenic ⌬slaA mutant strain was signifi- With the goal of directly testing the hypothesis that SlaA cantly impaired in ability to adhere to and kill human epithelial cells contributes to pathogenesis, we made a ⌬slaA isogenic mutant compared with the wild-type parental strain. The mutant strain strain from a wild-type serotype M3 strain and studied its role was less virulent for mice than the wild-type strain, and immuni- in GAS host–cell interaction and contribution to virulence in zation with purified SlaA significantly protected mice from invasive mouse and monkey models of infection. disease. Importantly, the mutant strain was significantly attenu- ated for colonization in a monkey model of pharyngitis. We Results conclude that transductional acquisition of the ability of a GAS Exogenously Presented SlaA Is Not Cytotoxic to Cultured Human strain to produce SlaA enhanced the spread and virulence of the Epithelial Cells. SlaA has PLA2 activity in vitro against several serotype M3 precursor strain. Hence, these studies identified a physiologically relevant substrates present in host cell mem- crucial molecular event underlying the evolution, rapid emergence, branes (14). Degradation of phospholipids by phospholipases and widespread dissemination of unusually severe human infec- can damage host membranes and decrease cell viability. To tions caused by a distinct bacterial clone. determine whether exogenous SlaA caused host-cell cytotoxic- MICROBIOLOGY ity, purified recombinant SlaA (rSlaA) was incubated with bacteria ͉ Group A Streptococcus ͉ Streptococcus pyogenes immortalized pharyngeal epithelial (D562) cells, and lactate dehydrogenase (LDH) released into the culture medium was lthough of tremendous concern to society and public health measured as an indicator of cell damage. No significant increase Aauthorities, the molecular events, epidemiological pro- in LDH activity was detected (data not shown). Similarly, rSlaA cesses, and host factors that contribute to rapid emergence of did not have a detrimental effect on host-cell morphology or new pathogenic bacterial clones are poorly understood (1, 2). membrane integrity, as assessed by transmission electron mi- Information about these processes is needed to develop a croscopy (Fig. 7, which is published as supporting information on predictive model of bacterial epidemics and new drugs, vaccines, the PNAS web site). These results indicated that exogenous and diagnostics. To better understand clone emergence and rSlaA alone does not produce a substantive cytotoxic effect on changes in disease frequency and severity, we have used genome- human epithelial cells grown in vitro. wide analysis methods to study group A Streptococcus (GAS), a model human pathogen (3–6). GAS causes infections ranging in SlaA Increases GAS Attachment to and Killing of Human Epithelial severity from relatively mild pharyngitis and skin infections to Cells. To determine whether SlaA contributes to pathogenesis, life-ending invasive diseases such as septicemia, necrotizing we made a ⌬slaA isogenic mutant strain from a wild-type fasciitis, and streptococcal toxic shock syndrome (7). serotype M3 strain (Figs. 8 and 9, which are published as Serotype M3 strains have been of particular interest, because supporting information on the PNAS web site). We tested the comprehensive population-based studies have shown that these hypothesis that SlaA production influenced GAS–host cell organisms cause a disproportionate number of severe invasive interaction by using D562 cells and normal human trancheo- disease infections, such as necrotizing fasciitis and death (8–11). bronchial epithelial (NHTBE) cells. In both cell types, signifi- Based on genome sequencing and molecular population genetic analysis of strains recovered over Ͼ60 years, we discovered that Author contributions: I.S. and J.M.M. designed research; I.S., M.J.N., P.S., S.D.B., and C.C.-B. acquisition of a bacteriophage encoding a new secreted phos- performed research; I.S., M.J.N., C.C.-B., and J.M.M. analyzed data; and I.S. and J.M.M. pholipase A2 (PLA2) named SlaA created a new clone that now wrote the paper. is responsible for the vast majority of human infections caused The authors declare no conflict of interest. by serotype M3 strains in many countries (3, 4, 12). SlaA is Abbreviations: GAS, Group A Streptococcus; PLA2, phospholipase A2; SlaA, streptococcal secreted extracellularly and is related to a potent toxin (textilo- phospholipase A; rSlaA, recombinant SlaA; NHTBE, normal human tracheobronchial epi- toxin) made by the Australian common brown snake, Pseudonaja thelial; SLO, streptolysin O; moi, multiplicity of infection. textilis (3, 13, 14). †Present address: Department of Pharmacology, University of North Carolina, Several lines of evidence suggest that SlaA is a GAS virulence Chapel Hill, NC 27599. factor. The slaA gene was not present in serotype M3 strains until ¶To whom correspondence should be addressed. E-mail: [email protected]. the mid-1980s, a time frame that correlated with the increase in © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607669103 PNAS ͉ October 24, 2006 ͉ vol. 103 ͉ no. 43 ͉ 16009–16014 Downloaded by guest on October 2, 2021 A A >100 B P=0.009 100 2 1 80 WT 0 IgG 60 -2 control 40 -4 B 20 -6 Attached bacteria per cell 10:1 30:1 300:1 Fold change in attachment -8 Anti-SlaA Ratio of GAS/D562 cells antibody 3.0 P=0.034 P=0.045 C D 12 70 C P=0.02 2.5 10 60 8 50 40 2.0 6 n.s. 30 1.5 4 20 Fold change in attachment % of dead cells 2 10 1.0 0 0 WT D562 cells NHTBE cells P=0.0005 +rSlaA WT D 35 WT 30 Fig. 2. Characterization of SlaA-dependent GAS–human cell interactions. (A) Adherence of the wild-type strain is dose-dependent, whereas adherence of the 25 P=0.004 ⌬slaA mutant strain is not. Experiments were performed with D562 cells as described but at the indicated mois. (B) Depletion of SlaA decreases GAS adher- 20 ence to D562 cells. SlaA was titrated with 10 ␮g of anti-SlaA antibody during 15 infection with wild-type MGAS315. Affinity-purified preimmune rabbit IgG at the same concentration was used as a control. (C) Addition of purified rSlaA to the 10 infection assay increased adherence of the ⌬slaA mutant strain. rSlaA (50 ␮g) was 5 added simultaneously with the wild-type and ⌬slaA mutant strains. n.s., not Attached bacteria per cell significant. (D) Production of SlaA significantly enhanced GAS killing of human 0 epithelial cells. D562 or NHTBE epithelial cells were cocultured with wild-type or D562 cells NHTBE cells the ⌬slaA isogenic mutant strain (moi ϭ 100:1, 3 h) and stained with trypan blue. The number of stained cells in 50 randomly chosen fields was counted by light Fig. 1. Production of SlaA significantly enhances adherence of GAS to microscopy, and the number of positive cells was expressed as percent of total human epithelial cells. D562 and NHTBE cells were infected with wild-type or cells in the field. P values were determined by t test of data for mutant vs. the ⌬slaA mutant strain (moi ϭ 100:1, 3 h), washed with PBS, fixed, and stained wild-type. NHTBE are primary cells and are much more susceptible to killing by with crystal violet. Photomicrographs for NHTBE cells are not shown, but the GAS than the immortalized D562 cells. results mirrored the D562 data. (A) Uninfected D562 cells. (B) D562 cells infected with wild-type strain MGAS315. (C) D562 cells infected with ⌬slaA isogenic mutant strain. (Magnification, ϫ40.) (D) Quantitative differences in host-cell adherence between the wild-type and ⌬slaA mutant strains by using type GAS, indicating that SlaA gained access to the host cell D562 cells (Left) and NHTBE cells (Right). The number of cell-associated cytoplasm (Fig. 3A). These results were confirmed with confocal bacteria was determined by counting GAS attached to 50 randomly selected microscopy (Fig. 3 B and C). Taken together, the data suggest human cells in five different microscope fields. The results are expressed as the that SlaA plays an important role in facilitating GAS adherence mean number of GAS per host cell. An enlarged version of Fig. 1 is presented to cultured epithelial cells, and that entry of SlaA into host cells as Fig. 10, which is published as supporting information on the PNAS web site. is required for cytotoxicity.

Load more

  6

Copy Link