Impact of Microorganisms

TWiM #195: Gingipain in the Alzheimer Brain

Podcast and Annotation Information
  • Annotation by Vasilios Loutrianakis, Rohit Cherian, Vedant Wasade, Mateo Pujol, Rebecca Seipelt-Thiemann, and Maggie Schlarman.
  • Podcast audio by TWiM: Listen to TWiM #195 Podcast
  • Podcast transcript by Otter.ai and edited by Harshita Sharma and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK, Vakulskas CA, Collingwood MA, Zhang L, Bode NM, Behlke MA, Dejene B, Cieniewicz B, Romano R, Lesch BJ, Gomez-Ospina N, Mantri S, Pavel-Dinu M, Weinberg KI, Porteus MH. 2019. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 25(2):249–254. doi: 10.1038/s41591-018-0326-x.
    • Dominy SS, Lynch C, Ermini F, Benedyk M, Marczyk A, Konradi A, Nguyen M, Haditsch U, Raha D, Griffin C, Holsinger LJ, Arastu-Kapur S, Kaba S, Lee A, Ryder MI, Potempa B, Mydel P, Hellvard A, Adamowicz K, Hasturk H, Walker GD, Reynolds EC, Faull RLM, Curtis MA, Dragunow M, Potempa J. 2019. Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Sci Adv.5(1):eaau3333. doi: 10.1126/sciadv.aau3333.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:35 minutes

The Most Interesting Things (according to students)

  • The discovery of Cas9 orthologs from bacteria that don’t infect us (not Staph or Strep Cas9). The whole paper discusses the negatives of us being immune to the benefits of Cas9, and actually the detriments, healthwise, that could arise because we already have antibodies against Streptococcus and Staphylococcus already.
  • The importance of not introducing “things” that could have large immune system responses, as seen with Jesse Gelsinger, who died of viral gene therapy. An immune reaction attacked his own organs and killed him due to receiving too much virus during the procedure. This is why a lot of blood transfusions or other donations also come with something that suppresses your immune response, like Benadryl, to prevent your body from reacting to the new foreign substance.
  • At the end of the Podcast, there was a brief moment when Dr. Racaniello suggested we could possibly ablate all the major antigenic sites of the two Cas9 proteins and hope it does not mess up the protein.  This would be such an interesting follow up to the paper because one would imagine that because so many of us are immune to both of these orthologs that there would be many antigenic sites and the protein would denature or deform completely, but then again, we’re not sure.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1. 

1.2. Main paper; discussion starts at 21:25 minutes

The Most Interesting Things (according to students)

  • Gingipains, which come from P. gingivalis, can be found not only in saliva, but also in the cerebral spinal fluid (CSF). This sets up the whole paper because, as they mention, any student of microbiology knows and appreciates that the CSF is a window to neurological infections of the brain and the neuronal system. This means that good oral hygiene reduces Alzheimer’s risk, what other good hygiene practices prevent other seemingly unrelated diseases?
  • The usefulness of the protein inhibitors and the efficacy of gingipain inhibitors seems super promising because it’s also a somewhat new idea in microbiology, limiting the usefulness of virulence factors rather than the bacteria itself, and introducing the possibility that antimicrobial therapy could be a treatment for Alzheimer’s Disease.

“Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer’s disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer’s patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1–42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1–42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer’s disease.” (Dominy et al. 2019, no changes)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.
  • Fundamental Statement 26 (ASM_26) :Humans leverage microbes and their products to address problems and improve quality of life.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, a minority have a detrimental impact on their host.
  • Fundamental Statement (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the function of specific components in CRISPR/Cas9 gene editing.
  • Recall suggested ways to combat preexisting immunity to CRISPR/Cas 9 gene therapy.
 S L
  • Predict how changes or alterations to the CRISPR technology components would affect the outcome, and vice versa.
 S H
  • Recall the roles and/or uses of proteins in the study (Tau, ubiquitin, proteases, amyloid beta).
  • Recognize the source of  P. gingivalis in the human body.
  • Identify the evidence linking P. gingivalis to Alzheimer’s disease.
 M L
  • Propose therapeutic approaches to target P. gingivalis and help treat Alzheimer’s disease.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • CRISPR/Cas9 (3:05 –  20:10): A repurposed defense system from bacteria that allows one to modify DNA sequences specifically. CRISPR is a short sequence used to target any gene and Cas9 is an endonuclease that cuts the target.
  • Western Blot (8:18–11:07): A technique used to detect and quantity proteins in a sample such as the human cord blood sample of mothers we see in the snippet paper, where they wanted to see the prevalence of the Cas9 protein of both Strep Cas9 and Staph Cas9 in the blood, and the researchers found incredibly high percentages of both Cas9 proteins. 

4.2. Main Paper

  • Immunohistochemistry (38:34–41:36): This detection method is used commonly to detect the presence of proteins in their tissue or cell location.  It uses antibodies to specific proteins and can be a color or fluorescent signal.  Here, brain tissue from Alzheimer’s patients was stained with antibodies targeting gingipains (bacterial proteases), tau (a hallmark of Alzheimer’s), and ubiquitin (a marker of protein degradation). This revealed a strong correlation between gingipain presence and tau pathology, suggesting that P. gingivalis may play a role in Alzheimer’s neurodegeneration.
  • PCR (39:23–41:46): Polymerase chain reaction (PCR) is a molecular detection method using primers, heat-stable DNA polymerase and a DNA template.  It is used to amplify copies of DNA segments very specifically.  Here, it is used to detect P. gingivalis DNA in saliva and cerebrospinal fluid.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Counteractions to Immunity (17:07–17:48): Because immunity is such a problem in gene therapy, one idea the two speakers discuss in counteracting our immune response is giving substances to suppress it, such as benadryl or something similar, to make sure that you do not react negatively to foreign material being introduced to you (Cas9 is foreign material in this case).  Another way to counteract immunity is to pass the Cas9 proteins through a fluorescent activated cell sorter (FACS) machine and use flow cytometry to sort Cas9 positive and Cas9 negative cells.  Selecting for the Cas9 negative cells would limit immune responses to Cas9 as an antigen.
  • Viral Gene Therapy (19:53–20:31): Treatment that uses viruses to deliver genetic material to cells and the baseline for modern CRISPR/Cas9. The paper discusses how controversial this method has been in the past, especially twenty years ago with one patient who died as a result of a massive immune reaction likely due to a viral dosage that was too high. This had profound effects on the whole field and is part of the reason for hesitation with CRISPR/Cas9 today.

5.2. Main Paper

  • Koch’s Postulates (23:17–1:00:51): Koch’s postulates are criteria used to evaluate evidence for a link between a microorganism and a disease. P. gingivalis is present in Alzheimer patient brains, and in animal models, it leads to neurodegeneration, when inhibited it prevents symptoms.
  • Virulence Factors (24:33–38:34): Molecules produced by pathogens that fight against one’s immune system to have a more efficient ability to cause disease. These can be toxins, enzymes, adhesion factors and more. Gingipains are the proteases that break down host proteins, the main virulence factor P. gingivalis uses.
  • Creation of Inhibitors (41:46–57:04): In order to combat the gingipains, they created small molecule inhibitors specific to the makeup of the gingipains.  Many were tried, for example since gingipains are cysteine proteases, they made an inhibitor that knocks down disulfide bridges in cysteine. Or, they made Arginine vs Lysine inhibitors because those were the two types of gingipains.  Remarkably, they found that when inhibitors were injected in mice, neurodegeneration slowed/stopped.

6. Podcast Questions

  1. What is the primary role of Cas9 in CRISPR gene editing?
    1. It guides the complex to specific sequences
    2. It cleaves DNA at the targeted locations
    3. It induces homologous recombination of DNA
    4. It replaces mutated DNA with wildtype DNA
  2. Which of the following is NOT a proposed strategy to overcome pre-existing immunity to Cas9?
    1. Using Cas9 from bacteria that do not commonly infect humans.
    2. Suppressing the immune system with immunosuppressive medications.
    3. Increasing the dosage of Cas9 to overpower immune responses.
    4. Modifying Cas9 epitopes to eliminate or reduce immune recognition.
  3. If you wanted to use gene editing in Streptococcus pyogenes, which CRISPR/Cas9 component would not be necessary to add from an external source, and why?
    1. The CRISPR protein; the protein is part of DNA repair.
    2. The palindromic repeat; repeats are species-specific.
    3. The guide RNA; it is produced by random transcription.
    4. The endonuclease; S. pyogenes already has this enzyme.
  4. If a researcher accidentally provided the incorrect guide RNA in CRISPR/Cas9-based gene editing what effect would this have?
    1. CRISPR/Cas9 would cleave a different DNA target.
    2. CRISPR/Cas9 would not be able to target DNA at all.
    3. CRISPR/Cas9 would target RNA instead of DNA.
    4. CRISPR/Cas9 would target DNA, but not cleave it.
  5. Which molecule functions in clearing damaged proteins?
    1. Tau
    2. Gingipain
    3. Beta amyloid
    4. Ubiquitin
  6. Where is Porphyromonas gingivalis commonly found in the human body?
    1. Gut microbiome
    2. Eye microbiome
    3. Oral microbiome
    4. Skin microbiome
  7. What evidence supports a causal role for P. gingivalis in Alzheimer’s disease?
    1. Antibiotics targeting P. gingivalis have been shown to completely cure Alzheimer’s Disease.
    2. Mice infected with P. gingivalis developed increased amyloid-beta plaques and cognitive impairment.
    3. People with Alzheimer’s disease have no detectable P. gingivalis anywhere on their bodies.
    4. Patients treated for P. gingivalis infections have a significantly higher risk of developing Alzheimer’s.
    5. The presence of viral RNA in Alzheimer’s patients’ cerebrospinal fluid this virus causes it.
    6. The discovery of tau aggregates in all neurodegenerative diseases, one of which is Alzheimer’s.
    7. P. gingivalis DNA and gingipains were found in affected brains and correlated with disease markers.
    8. The observation that only elderly individuals, and no your individuals experience Alzheimer’s disease
  8. Gingipains are proteases that are known virulence factors produced by P. gingivalis.  The investigators synthesized small molecule inhibitors that they successfully tested against P. gingivalis. What would be an effective common place to include these inhibitors for treatment purposes?
    1. Toothpaste
    2. Sunscreen
    3. Detergent
    4. Eye drops

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (extended data Figure 2) and one from the main paper (Figure 1 C-H).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify the reasoning for the researchers’ experimental choices.
  • Identify the positive and negative controls in the experiment, as well as the features of the ELISA graph.
  • Analyze ELISA experimental results for specific conditions.
  • Determine whether these data support or do not support the hypothesis that there is pre-existing immunity to Cas9 proteins in human subjects.
Experimental Background (Charlesworth et al., Extended Data Figure 2)

CRISPR-Cas9 is a genome-editing technology that enables precise modification of DNA and holds promise for treating genetic diseases. However, as Cas9 proteins used in this system are derived from common human pathogens such as Staphylococcus aureus (SaCas9) and Streptococcus pyogenes (SpCas9), there is concern that people may already have immune responses against them. Pre-existing adaptive immunity could compromise the safety and effectiveness of CRISPR-based therapies, especially those delivered directly into the body.  In this study, Charlesworth et al. (2019) investigated whether healthy individuals (noted by individual symbols) harbor preexisting antibodies against SaCas9 and SpCas9 using an ELISA (enzyme-linked immunosorbent assay) to measure the presence of Cas9-specific antibodies in human serum. Serum antibody different dilutions (1:10, 1:50, 1:100, and 1:1000) were tested against Cas9 proteins, along with tetanus toxoid (TT), which is commonly vaccinated against in the population, and human albumin (HA).

7.1.2. Questions

  1. The positive control for this experiment is _____ and the negative control for this experiment is ______.
    1. TT; HA
    2. SaCas9; SpCas9
    3. HA; TT
    4. SpCas9; SaCas9
  2. What do the individual symbols for each protein/dilution, such as the circles for TT in the top left panel, indicate?
    1. Each symbol is a technical replicate for a single person’s serum.
    2. Each symbol is a biological replicate for a group of sera.
    3. Each symbol is an ELISA result for a specific person’s serum.
  3. Why are the researchers focusing on detecting immune reactivity/antibody binding against SaCas9 and SpCas9? Pick the best choice.
    1. These are the most dangerous Cas9 proteins found in bacteria that can infect humans.
    2. To identify the most effective evasion-inducing antibodies for specific virulence factors.
    3. To determine whether gene editing poses a safety risk due to immune reactivity in humans.
    4. To evaluate diverging evolution between S. aureus and S. pyogenes Cas9 proteins.
  4. What does a high OD₄₅₀ reading in the ELISA assay indicate?
    1. A lack of protein-antibody binding in the sample
    2. A strong antibody level to the target antigen
    3. High T cell activation against the target antigen
    4. Protein degradation took place in the ELISA wells
  5. Which of the following best explains the trend you would expect to observe for the positive immune reaction as serum is increasingly diluted, such as from 1:10 to 1:1000?
    1. OD decreases with dilution, but remains detectable
    2. OD increases with dilution, showing more specificity
    3. OD is constant across all samples due to cross-reactivity
    4. OD is consistently low, showing no immune reactivity
  6. Which antigen showed the highest average antibody reactivity/binding across all serum dilutions?
    1. Human albumin
    2. SpCas9
    3. SaCas9
    4. Tetanus toxoid
  7. Do these data support or not support that antibodies to Cas9 proteins used commonly in gene editing exist in the serum of these individuals? What is your evidence?
    1. Yes, average OD values for SaCas9 and SpCas9 are above the negative control for each serum dilution.
    2. No, average OD values for SaCas9 and SpCas9 are less than the positive control for each serum dilution
    3. Yes, average OD values for SaCas9 and SpCas9 are both detectable and increase with each serum dilution.
    4. No, average OD values for SaCas9 and SpCas9 are undetectable at all serum dilutions.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify features of the experimental methods related to the displayed results.
  • Analyze gingipain staining intensity graphs to determine any differences between Alzheimer’s patients and non-demented controls.
  • Evaluate the strength of the evidence correlating P. gingivalis infection to Alzheimer’s pathology based on the immunohistochemical quantification.
Experimental Background (Dominy et al., Figure 1 C-H)

Alzheimer’s Disease is a debilitating neurological disease that is known to have many contributing factors. Dominy et al. (2019) investigated the possibility that infection with a bacterium known to inhabit periodontal plaques and the oral microbiome might play a role in Alzheimer’s Disease.  They quantified the presence of several things in both Alzheimer’s Disease patients and age/sex-matched controls.  They quantified P. gingivalis cysteine protease virulence factors RgpB (arginine gingipain B) and Kgp (lysine gingipain), which are essential for P. gingivalis survival and pathogenicity as well asTau and Ubiquitin.  High levels of Tau and Ubiquitin are hallmarks of  Alzheimer’s Disease.  The method they use is immunohistochemistry on tissue microarrays (TMA NVD005) from the middle temporal gyrus using specific antibodies CAB101 for RgpB and CAB102 for Kgp.9-part infographic of control and experiment results affect on RgpB, Tau, and Kgp load.Figure 1. “Gingipain IR in brain correlates with AD diagnosis and pathology. (A and B) Representative TMA NVD005 containing brain tissue cores from the MTG of AD patients and controls probed for RgpB (A) and Kgp (B) with antibodies CAB101 and CAB102, respectively. Higher magnification of representative tissue cores reveals higher neuronal RgpB-IR and Kgp-IR in AD tissue cores than in control cores. (C) RgpB-IR and (D) Kgp-IR data from TMAs NVD005 and NVD003 show significantly higher load in AD brain compared to controls. Mann-Whitney test, ***P < 0.0001; presented as geometric mean ± 95% confidence interval, n = 99 (C) and n = 104 (D). (E and F) Tau load correlates to RgpB load (Spearman r = 0.674, P < 0.0001, n = 84) (E) and Kgp load (Spearman r = 0.563, P < 0.0001, n = 89) (F). Blue, control; red, AD. (G and H) Ubiquitin load, a marker of AD pathology, correlates to RgpB load (blue, control; red, AD; Spearman r = 0.786, P < 0.0001, n = 99) (G) and Kgp load (Spearman r = 0.572, P < 0.0001, n = 104) (H). (I) RgpB load correlates with Kgp load (Spearman r = 0.610, P < 0.0001, n = 99).” (Dominy et al., 2019)

7.2.2. Questions

  1.  The researchers compare the levels of lysine gingipain in Alzheimer’s Disease patients and controls in panel D.  Which color represents the data for Alzheimer’s Disease patients?
    1. blue
    2. red
  2. The researchers compare the levels of arginine gingipain B in Alzheimer’s Disease patients and controls in panel C.  Which antibody would they have used for this experiment?
    1. CAB101
    2. CAB102
    3. CAB103
    4. CAB104
  3. Arginine gingipain B load and lysine gingipain load in Alzheimer’s Disease patients and controls are quantified in panels C and D, respectively.  What can you conclude about these data?
    1. The data overlap for each instance, so there is no difference in Alzheimer’s Disease patients and controls.
    2. The data overlap, but the means are different and statistical measures indicate there is a difference.
    3. The data do not overlap, so Alzheimer’s Disease patients and controls are different in RgpB and Kgp.
    4. The data barely overlap, so Alzheimer’s Disease patients and controls are slightly different in RgpB and Kgp.
  4. Which panel allows us to evaluate the correlation of arginine gingipain B level and tau level?  What do those results indicate for the relationship? What is your evidence?
    1. Panel G; positive correlation; the slope of the best fit line increases and patients (blue) are found at the higher end while controls (red) are found at the lower end.
    2. Panel F; no correlation; the slope of the best fit line is small with patient (blue) and controls (red) found all along the entire distribution of the line length.
    3. Panel E; positive correlation; the slope of the best fit line increases and patient (red) are found at the higher end while  controls (blue) are found at the lower end.
    4. Panel H; no correlation; the slope of the best fit line is small with Alzheimer’s Disease patient (red) and controls (blue) found all along the entire distribution of the line length.
  5. Which of the following best describes the conclusions about gingipain, tau, and ubiquitin levels in these experiment results?
    1. Gingipain levels are lower in AD patients and inversely related to tau and ubiquitin accumulation.
    2. Gingipain and ubiquitin levels do not significantly differ, but gingipain and tau levels correlate.
    3. Gingipain levels are higher in AD patients and positively correlate with tau and ubiquitin load.
    4. Gingipains are only found in control brain tissues; tau and ubiquitin are present only in AD brain.

8. Paper Information and Licensing

8.1. Snippet paper

  • Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK, Vakulskas CA, Collingwood MA, Zhang L, Bode NM, Behlke MA, Dejene B, Cieniewicz B, Romano R, Lesch BJ, Gomez-Ospina N, Mantri S, Pavel-Dinu M, Weinberg KI, Porteus MH. 2019. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 25(2):249–254. doi: 10.1038/s41591-018-0326-x.
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. A version is available on PubMed at: https://pmc.ncbi.nlm.nih.gov/articles/PMC7199589/

8.2. Main paper

  • Dominy SS, Lynch C, Ermini F, Benedyk M, Marczyk A, Konradi A, Nguyen M, Haditsch U, Raha D, Griffin C, Holsinger LJ, Arastu-Kapur S, Kaba S, Lee A, Ryder MI, Potempa B, Mydel P, Hellvard A, Adamowicz K, Hasturk H, Walker GD, Reynolds EC, Faull RLM, Curtis MA, Dragunow M, Potempa J. 2019.Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Sci Adv.5(1):eaau3333. doi: 10.1126/sciadv.aau3333.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journal’s web page.

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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