Dataset Information

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

ABSTRACT: Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co-culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air-liquid interface to allow an in-depth evaluation of a simulated colonization state. Exposure to wild-type, S. aureus bacteria or conditioned media killed airway epithelial cells within 1?day, while mutant S. aureus strains lacking alpha-toxin (hla) persisted on viable cells for at least 2?days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real-time polymerase chain reaction of known virulence factors showed the expression profile of S. aureus grown in co-culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co-culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.

SUBMITTER: Kiedrowski MR

PROVIDER: S-EPMC4840028 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

Kiedrowski Megan R MR Paharik Alexandra E AE Ackermann Laynez W LW Shelton Annie U AU Singh Sachinkumar B SB Starner Timothy D TD Horswill Alexander R AR

Cellular microbiology 20160112 5

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co-culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air-liquid interface to allow ...[more]

PMID: 26566259

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Project description:Staphylococcus aureus nasal colonization is a risk factor for infection. A large proportion of the population are identified as potential S. aureus carriers yet we only partially understand the repertoire of genetic factors that promote long-term nasal colonization. Here we present a murine model of nasopharyngeal colonization that requires a low S. aureus inoculum and is amenable to experimental evolution approaches. We used this model to experimentally evolve S. aureus using successive passages in the nasopharynx to identify those genetic loci under selection. After 3 cycles of colonization, mutations were identified in mannitol, sorbitol, arginine, nitrite and lactate metabolism genes promoting key pathways in nasal colonization. Stress responses were identified as being under selective pressure, with mutations in DNA repair genes including dnaJ and recF and key stress response genes clpL, rpoB and ahpF. Peptidoglycan synthesis pathway genes also revealed mutations indicating potential selection for alteration of the cell surface. The murine model used here is versatile to question colonization, persistence and evolution studies. We studied the human pathogen Staphylococcus aureus in our search to determine factors that contribute to its ability to live in the human nose and throat. The anterior nares and nasopharynx are considered primary habitats but we do not understand how the pathogen adapts as it moves from one person to the next. We first determined sustained survival of the pathogen over multiple days in the nasopharynx that might act as a good model for human persistence due to the low numbers of bacteria needed for it to establish. By using successive rounds of colonization of the nasopharynx across different mice we revealed that multiple genetic changes in the S. aureus occurred. These changes were found in genes associated with the cell surface and metabolism and might indicate adaptation to the niche. One gene showed an accumulation of multiple mutations supporting a key contribution in adaptation but the role of the protein it encodes is not yet known. The contribution of these genes and genetic changes are unclear but indicate an area for future research to better understand how this common human pathogen is so successful at human colonization and survival.

Project description:Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.

Dataset Information

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

Publications

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia.

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