Lead from historical gasoline use continues to affect air quality in urban London almost 20 years after its phase-out.
Image credit: Eléonore Resongles and Volker Dietze.
Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved June 13, 2014 (received for review December 12, 2013)
Significance
Increasing the integrity of the bacterial envelope is necessary to allow the successful survival of bacterial pathogens within the host and allow them to counteract damage caused by membrane-targeting antibiotics. We demonstrate that components of a clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) system, a prokaryotic defense against viruses and foreign nucleic acid, act to regulate the permeability of the bacterial envelope, ultimately providing these cells with the capability to resist membrane damage caused by antibiotics. This regulation further allows bacteria to resist detection by multiple host receptors to promote virulence. Overall, this study demonstrates the breadth of function of CRISPR-Cas systems in regulation, antibiotic resistance, innate immune evasion, and virulence.
Abstract
Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
Footnotes
↵1B.A.N. and M.R.S. contributed equally to this work.
↵2Present address: Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305.
↵3Present address: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840.
↵4Present address: Department of Wound Infections, Walter Reed Army Institute of Research, Silver Spring, MD 20910.
- ↵5To whom correspondence should be addressed. Email: david.weiss{at}emory.edu.
Author contributions: T.R.S. and D.S.W. designed research; T.R.S., B.A.N., M.R.S., R.L., J.Z., C.-Y.C., H.K.R., A.C.L., C.L.J., P.Z., and H.P.E. performed research; H.L. and D.M. contributed new reagents/analytic tools; T.R.S., B.A.N., M.R.S., R.L., J.Z., C.-Y.C., H.K.R., C.L.J., P.Z., H.P.E., and D.S.W. analyzed data; B.A.N. performed the immunofluorescence staining and imaging; M.R.S. developed and executed the polymyxin B screen; R.L. and H.P.E. performed experiments with C. jejuni; J.Z. and P.Z. isolated and analyzed the lipid A; C.-Y.C. performed surface charge analysis; H.K.R. performed susceptibility assays; A.C.L. generated the FTN_1254 and FTN_0109 mutants; C.L.J. made fundamental contributions to experimental direction; T.R.S. performed all other experiments; and T.R.S. and D.S.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1323025111/-/DCSupplemental.
Envelope enhancement and immune evasion by Cas9
Sign up for Article Alerts
Jump to section
You May Also be Interested in
Winter rainfall in sub-Saharan Africa limits the size of painted lady butterfly migrations to Europe in the spring.
Image credit: Oriol Massana Valeriano (photographer).
Many languages in which unique medicinal knowledge exists are at risk of extinction, and indigenous language loss may result in lost medicinal knowledge.
Image credit: Pixabay.
By borrowing the machinery of life from broken cells, researchers are producing novel proteins, cheap lab reagents, and just-add-water vaccines.
Image credit: Dave Cutler (artist).
When playing the Ultimatum Game, chimps and bonobos give away a bigger share if they see the other animal has access to an alternative offer.
Image credit: Science Source/Kenneth W. Fink.