Heavy metals like iron, copper, manganese and zinc can be toxic in large amounts. But in low concentrations, they are essential micronutrients for humans as well as bacteria. As part of the defense against invasive microbes, our immune system exploits this property by starving bacteria of the metals they need to survive. One way of doing this is with calprotectin, an immune defense protein produced by neutrophils at the site of infections. Calprotectin depletes the local environment of metal nutrients, and can block Borrelia burgdorferi growth. The mechanism of how this works has now been explained in a newly published study, funded by GLA and led by Dr. Valeria Culotta.

Calprotectin was found in the dermal and epidermal layers of erythema migrans rashes of early Lyme disease patients. It is believed that epithelial keratinocytes in the skin produce calprotectin, which is more commonly associated with neutrophils, immune cells that are absent in this particular site. Surprisingly, calprotectin blocked B. burgdorferi growth not by depleting metals, as would be expected. Instead, it directly bound to the bacteria and changed their shape from spirochetes into a cyst-like form, when immersed in water. This differs from calprotectin’s mechanism of blocking another bacteria, E. coli, in which metal sequestration is essential to blocking bacterial replication. These findings suggest a unique new pathway that could be explored for a potential therapeutic that is specific to B. burgdorferi.

Borrelia burgdorferi (Bb) enters the human body via the bite of an infected Ixodes tick. Lyme disease symptoms arise from the dissemination of Bb from the initial bite site to the distal tissues such as the heart, the joints, or the nervous system. We identified a novel Bb infectivity gene, bbk13, that is important for distal tissue colonization in a mouse model of infection. Detailed characterization of the infection kinetics showed that there is a critical Bb population expansion that occurs at the skin during early infection and that bbk13 is important for this process. This study highlights the significance of early spirochete expansion in the skin towards driving downstream events that lead to distal tissue colonization. Therapeutic approaches that limit the early, localized expansion in the skin may prevent the onset of disseminated Lyme disease symptoms.

Published in American Society for Microbiology: https://iai.asm.org/content/87/5/e00887-18
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30782856

The pathogenic spirochete Borrelia burgdorferi senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in Ixodes ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation.

Published in American Society for Microbiology–Journal of Bacteriology: https://jb.asm.org/content/201/4/e00582-18
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30478087

Dr. Alan Barbour and his team have analyzed the mitochondrial DNA from wild white-footed mice, Peromyscus leucopus. These mice are the natural host for Lyme and other tickborne bacteria, and serve as a reservoir from which ticks acquire pathogens. However, the mice themselves do not become sick despite being infected. This work focuses on mitochondria, the organelle responsible for generating energy in living cells. By sequencing and analyzing mitochondrial DNA and its transcription, Dr. Barbour’s group has provided the groundwork to determine how mice can tolerate infection, compared to lab mice and humans who do suffer symptoms. In addition, because mitochondrial DNA is inherited directly from the mother, genetic studies to understand Peromyscus subspecies geographic distribution and vectorborne disease dynamics can now be undertaken.

Published in Nature: https://www.nature.com/articles/s41598-019-54389-3
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31772306 

Persister forms of Borrelia burgdorferi, the bacterium that causes Lyme disease, are dormant or slow-growing, and tolerant of antibiotic treatment. It’s not clear yet whether persister bacteria, immune dysfunction, or some combination of the two is responsible for post-treatment Lyme disease syndrome (PTLDS), in which patients treated with antibiotics continue to suffer symptoms.

The search for novel compounds to kill persister bacteria has led to the discovery that essential oils (EOs), aromatic compounds produced by plants, may be promising. In an article published in Antibiotics, a peer-reviewed journal, scientists led by Dr. Ying Zhang of Johns Hopkins University identified 10 EOs that have strong activity against stationary phase B. burgdorferi at a low concentration of 0.1%. The study, underwritten by GLA, found that of the 10 EOs, those of garlic, allspice, and Palmarosa were active at even at 0.05% concentration. In addition, cinnamaldehyde, a major ingredient isolated from cinnamon bark, was active against both stationary phase bacteria as well as replicating B. burgdorferi at a 0.02% concentration.

A stringent test of antimicrobial activity against stationary phase bacteria is the capacity to block subcultured bacteria from growing. This means that after killing bacteria in culture with the inhibitor, a small amount of that culture is transferred to fresh growth media that lacks the inhibitor. Any regrowth indicates that the inhibitor did not completely kill all bacteria from the original culture. Under these conditions, only garlic and cinnamaldehyde were effective against the regrowth of B. burgdorferi spirochetes subcultured for 21 days.

These results indicate that certain EOs or their ingredients are potent in eliminating persister B. burgdorferi, and should be studied in greater depth to analyze their utility as potential treatments.

Lymphatic vessels collect interstitial fluid that has extravasated from blood vessels and return it to the circulatory system. Another important function of the lymphatic network is to facilitate immune cell migration and antigen transport from the periphery to draining lymph nodes. This migration plays a crucial role in immune surveillance, initiation of immune responses and tolerance.

Published in Frontiers in : https://www.frontiersin.org/articles/10.3389/fimmu.2019.01168/full
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31191539

Although most patients with Lyme disease can be cured with a 2-4 week antibiotic therapy, about 10-20% of patients continue to suffer prolonged persistent symptoms, a condition called post-treatment Lyme disease syndrome (PTLDS). The cause for PTLDS is unclear and hotly debated. B. burgdorferi develops morphological variants under stress conditions but their significance is not clear. Here we isolated the biofilm-like microcolony (MC) and planktonic (spirochetal form and round body) (SP) variant forms from the stationary phase culture and showed that the MC and SP were not only more tolerant to the current Lyme antibiotics but also caused more severe arthritis in mice than the log phase spirochete form (LOG). We propose to divide the persistent Lyme disease into two categories: (1) early development of persistent disease from inoculation with persister/biofilm at the beginning of infection introduced by tick bites, or Type I persistent disease (i.e., PTLDS); and (2) late development of persistent disease due to initial infection not being diagnosed or treated in time such that the infection develops into late persistent disease, or Type II persistent disease. Importantly, we show that the murine infection caused by LOG could be eradicated by ceftriaxone whereas the persistent infection established with MC could not be eradicated by doxycycline (Doxy), ceftriaxone (CefT), or vancomycin (Van), or Doxy+CefT or Van+CefT, but could only be eradicated by the persister drug combination daptomycin+doxycycline+ceftriaxone. Our studies demonstrate that varying levels of persistence and pathologies of Borrelia infection can be established with heterogeneous inocula with different morphologies and have different treatment responses. These observations may have broad implications for understanding pathogenesis and treatment of not only persistent Lyme disease but also other persistent infections in general and call for treatment of persistent infections with persister drug combination regimens that are more effective than the current often single-antibiotic monotherapy treatment.

Published in Discovery Medicine: http://www.discoverymedicine.com/Jie-Feng/2019/03/persister-biofilm-microcolony-borrelia-burgdorferi-causes-severe-lyme-arthritis-in-mouse-model/ 

Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. The goal of this study was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention.

Published in Frontiers in Microbiology: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00690/full
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31057493

This team, based at University of Connecticut, studied genes controlled by the RpoS pathway, the regulatory system that facilitates transmission and mammalian infection by Borrelia burgdorferi. They were particularly interested in whether RpoS-controlled genes are needed for chronic infection. They were able to show, contrary to current hypotheses, that OspA was expressed on spirochetes delivered via tick, then quickly turned off via RpoS repression. OspA remains repressed until subsequent acquisition by feeding larvae. RpoS is known to be required for the early stages of infection. But it’s been hard to study RpoS during chronic infection, because mutant strains of bacteria that lack RpoS aren’t virulent. Using a creative approach, the team confirmed that expression of RpoS is required throughout infection. In support of these findings, they identified an RpoS-regulated protein that imports peptides, OppA5, that are required to maintain bacterial persistence during chronic infection. OppA5 has thus been deemed a “persistence” gene, validating the hypothesis that a specific subset of RpoS-regulated genes are essential for maintenance of chronic infection.

Published in Frontiers in Microbiology: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719511/
PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31507550