skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 5:00 PM ET until 11:00 PM ET on Friday, June 21 due to maintenance. We apologize for the inconvenience.


This content will become publicly available on October 17, 2024

Title: Escherichia coli cells are primed for survival before lethal antibiotic stress
ABSTRACT

Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress (“primed cells”) resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation.

IMPORTANCE

Antibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information (“memory”) to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics.

 
more » « less
Award ID(s):
2240028 1922542
NSF-PAR ID:
10492420
Author(s) / Creator(s):
; ;
Editor(s):
Pagliara, Stefano
Publisher / Repository:
ASM
Date Published:
Journal Name:
Microbiology Spectrum
Volume:
11
Issue:
5
ISSN:
2165-0497
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    All the different forms of life on our planet – including animals, plants, fungi and bacteria – tend to grow, multiply and expand. This happens through a process called cell division, where one cell becomes two; two cells become four; four cells become eight; and so on. Each dividing cell passes on the same set of genetic instructions to its two daughter cells in the form of DNA. Its remaining contents, made up of a mixture of proteins, RNA and other chemicals, also get divided up equally between the two new cells. This division of cellular assets establishes a form of 'cellular memory', where daughter cells retain very similar properties to their ancestors, which helps them remain stable over time. Yet this memory can fade, and small changes in how a cell looks or acts can appear over many generations of cell division. This happens even when the exact same set of DNA-based genetic instructions have been passed down to daughter cells, confirming that other factors aside from DNA do influence cellular properties and can act to maintain them or introduce variation over time. Here, Vashistha, Kohram and Salman set out to understand how long cellular memory could be maintained in dividing E. coli bacteria. To do this, they created a technique to track cellular memory as it passed down from a single mother cell to two daughter cells over dozens of generations. Using this technique, Vashistha, Kohram and Salman found that some inherited elements, including cell size and the time cells took to divide, were maintained between mother and daughter cells for almost 10 generations. Other elements, such as the density of proteins inside each cell, started changing almost immediately after daughter cells were formed, and only remained similar for about two generations. These findings suggest that cellular memory may be long, but is not infinite, and that inheritance of non-genetic elements can help maintain cellular memory and reduce variation among new-born cells for considerable number of generations. Building on this research to achieve a better understanding of cellular memory may allow researchers to harness these insights to direct the evolution of different cellular properties over time. This could have a wide range of potential applications, such as designing new infection control measures for viruses or bacteria; enhancing our ability to grow working organs for tissue transplant; or improving the texture and consistency of cultured, lab-grown meat. 
    more » « less
  2. The specificity of T cells is that each T cell has only one T cell receptor (TCR). A T cell clone represents a collection of T cells with the same TCR sequence. Thus, the number of different T cell clones in an organism reflects the number of different T cell receptors (TCRs) that arise from recombination of the V(D)J gene segments during T cell development in the thymus. TCR diversity and more specifically, the clone abundance distribution, are important factors in immune functions. Specific recombination patterns occur more frequently than others while subsequent interactions between TCRs and self-antigens are known to trigger proliferation and sustain naive T cell survival. These processes are TCR-dependent, leading to clone-dependent thymic export and naive T cell proliferation rates. We describe the heterogeneous steady-state population of naive T cells (those that have not yet been antigenically triggered) by using a mean-field model of a regulated birth-death-immigration process. After accounting for random sampling, we investigate how TCR-dependent heterogeneities in immigration and proliferation rates affect the shape of clone abundance distributions (the number of different clones that are represented by a specific number of cells, or “clone counts”). By using reasonable physiological parameter values and fitting predicted clone counts to experimentally sampled clone abundances, we show that realistic levels of heterogeneity in immigration rates cause very little change to predicted clone-counts, but that modest heterogeneity in proliferation rates can generate the observed clone abundances. Our analysis provides constraints among physiological parameters that are necessary to yield predictions that qualitatively match the data. Assumptions of the model and potentially other important mechanistic factors are discussed. 
    more » « less
  3. The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2–4 μM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application. 
    more » « less
  4. ABSTRACT For insects that depend on one or more bacterial endosymbionts for survival, it is critical that these bacteria are faithfully transmitted between insect generations. Cicadas harbor two essential bacterial endosymbionts, “ Candidatus Sulcia muelleri” and “ Candidatus Hodgkinia cicadicola.” In some cicada species, Hodgkinia has fragmented into multiple distinct but interdependent cellular and genomic lineages that can differ in abundance by more than two orders of magnitude. This complexity presents a potential problem for the host cicada, because low-abundance but essential Hodgkinia lineages risk being lost during the symbiont transmission bottleneck from mother to egg. Here we show that all cicada eggs seem to receive the full complement of Hodgkinia lineages, and that in cicadas with more complex Hodgkinia this outcome is achieved by increasing the number of Hodgkinia cells transmitted by up to 6-fold. We further show that cicada species with varying Hodgkinia complexity do not visibly alter their transmission mechanism at the resolution of cell biological structures. Together these data suggest that a major cicada adaptation to changes in endosymbiont complexity is an increase in the number of Hodgkinia cells transmitted to each egg. We hypothesize that the requirement to increase the symbiont titer is one of the costs associated with Hodgkinia fragmentation. IMPORTANCE Sap-feeding insects critically rely on one or more bacteria or fungi to provide essential nutrients that are not available at sufficient levels in their diets. These microbes are passed between insect generations when the mother places a small packet of microbes into each of her eggs before it is laid. We have previously described an unusual lineage fragmentation process in a nutritional endosymbiotic bacterium of cicadas called Hodgkinia . In some cicadas, a single Hodgkinia lineage has split into numerous related lineages, each performing a subset of original function and therefore each required for normal host function. Here we test how this splitting process affects symbiont transmission to eggs. We find that cicadas dramatically increase the titer of Hodgkinia cells passed to each egg in response to lineage fragmentation, and we hypothesize that this increase in bacterial cell count is one of the major costs associated with endosymbiont fragmentation. 
    more » « less
  5. ABSTRACT Bacteria form complex multicellular structures on solid surfaces known as biofilms, which allow them to survive in harsh environments. A hallmark characteristic of mature biofilms is the high-level antibiotic tolerance (up to 1,000 times) compared with that of planktonic cells. Here, we report our new findings that biofilm cells are not always more tolerant to antibiotics than planktonic cells in the same culture. Specifically, Escherichia coli RP437 exhibited a dynamic change in antibiotic susceptibility during its early-stage biofilm formation. This phenomenon was not strain specific. Upon initial attachment, surface-associated cells became more sensitive to antibiotics than planktonic cells. By controlling the cell adhesion and cluster size using patterned E. coli biofilms, cells involved in the interaction between cell clusters during microcolony formation were found to be more susceptible to ampicillin than cells within clusters, suggesting a role of cell-cell interactions in biofilm-associated antibiotic tolerance. After this stage, biofilm cells became less susceptible to ampicillin and ofloxacin than planktonic cells. However, when the cells were detached by sonication, both antibiotics were more effective in killing the detached biofilm cells than the planktonic cells. Collectively, these results indicate that biofilm formation involves active cellular activities in adaption to the attached life form and interactions between cell clusters to build the complex structure of a biofilm, which can render these cells more susceptible to antibiotics. These findings shed new light on bacterial antibiotic susceptibility during biofilm formation and can guide the design of better antifouling surfaces, e.g., those with micron-scale topographic structures to interrupt cell-cell interactions. IMPORTANCE Mature biofilms are known for their high-level tolerance to antibiotics; however, antibiotic susceptibility of sessile cells during early-stage biofilm formation is not well understood. In this study, we aim to fill this knowledge gap by following bacterial antibiotic susceptibility during early-stage biofilm formation. We found that the attached cells have a dynamic change in antibiotic susceptibility, and during certain phases, they can be more sensitive to antibiotics than planktonic counterparts in the same culture. Using surface chemistry-controlled patterned biofilm formation, cell-surface and cell-cell interactions were found to affect the antibiotic susceptibility of attached cells. Collectively, these findings provide new insights into biofilm physiology and reveal how adaptation to the attached life form may influence antibiotic susceptibility of bacterial cells. 
    more » « less