Host and fungal factors both contribute to cryptococcosis-associated hyperammonemia (cryptammonia)

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are both known urease producers and have the potential to cause hyperammonemia. We hypothesized that the risk of hyperammonemia is increased by renal failure, burden of cryptococcal infection, and fungal strain characteristics. We performed a retrospective review of plasma ammonia levels in patients with cryptococcal infections. Risk factors for hyperammonemia were statistically compared between patients with and without hyperammonemia (>53 µmol/L). Cryptococcal cells from three patients included in the study were recovered from our biorepository. Strain characteristics including urease activity, ammonia production, growth curves, microscopy, melanin production, and M13 molecular typing were analyzed and compared with a wild-type (WT) C. neoformans strain. We included 29 patients, of whom 37.9% had hyperammonemia, 59% had disseminated cryptococcal infection (DCI), and 41% had isolated central nervous system infection. Thirty-eight percent of patients had renal failure and 28% had liver disease. Renal failure was associated with 4.4 times (95% confidence interval [CI] 1.5, 13.0) higher risk of hyperammonemia. This risk was higher in DCIs (RR 6.2, 95% CI 1.0, 40.2) versus isolated cryptococcal meningitis (RR 2.5, 95% CI, 0.40, 16.0). Liver disease and cryptococcal titers were not associated with hyperammonemia. C. neoformans from one patient with extreme hyperammonemia demonstrated a 4- to 5-fold increase in extracellular urease activity, slow growth, enlarged cell size phenotypes, and diminished virulence factors. Hyperammonemia was strongly associated with renal failure in individuals with DCI, surpassing associations with liver failure or cryptococcal titers. However, profound hyperammonemia in one patient was attributable to high levels of urease secretion unique to that cryptococcal strain. Prospective studies are crucial to exploring the significance of this association. IMPORTANCE Cryptococcus produces and secretes the urease enzyme to facilitate its colonization of the host. Urease breaks down urea into ammonia, overwhelming the liver’s detoxification process and leading to hyperammonemia in some hosts. This underrecognized complication exacerbates organ dysfunction alongside the infection. Our study investigated this intricate relationship, uncovering a strong association between the development of hyperammonemia and renal failure in patients with cryptococcal infections, particularly those with disseminated infections. We also explore mechanisms underlying increased urease activity, specifically in strains associated with extreme hyperammonemia. Our discoveries provide a foundation for advancing research into cryptococcal metabolism and identifying therapeutic targets to enhance patient outcomes.


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Reviewer #1 (Comments for the Author): The paper by Baker et al. evaluated the association of hyperammonemia, kidney failure and cryptococcosis.They found that patients with renal failure and cryptococcosis had a much higher risk of hyperammonemia, particularly those patients with disseminated cryptococcosis (presence of cryptococcal cells in the bloodstream).Although small, this retrospective study is well conducted and well analyzed.Statistic is appropriate.The results are well illustrated, well presented, and well discussed.The methods are clearly outlined.Few issues are below.
1.It is unclear whether the isolates from the 29 patients were Cryptococcus neoformans or Cryptococcus gattii.2. It is unclear why only few clinical cryptococcal isolates were characterized for urease activity and other fungal characteristics.After all, the authors should have access to all isolates from the 29 patients (either from blood or/and CSF).
Reviewer #2 (Comments for the Author): In this study, the authors investigated the risk factors for hyperammonemia in patients with Cryptococcus neoformans and gattii infections.A retrospective analysis of 29 patients revealed that 37.9% had hyperammonemia, with renal failure showing a significant association, increasing the risk four-fold.While disseminated cryptococcal infections had a higher risk of hyperammonemia compared to isolated meningitis, a unique strain with increased urease activity was identified in one patient with extreme hyperammonemia.Urease inhibition with an FDA-approved inhibitor was able to damper urease activity and proliferation by all strains.This manuscript is well written and and the experiments have been thoroughly planned and executed.The subject matter sheds light on an underappreciated complication that should garner strong general interest for the spectrum reader base.The most major limitation, being a low sample size, has been addressed.I only have minor concerns and suggestions, listed below, that should be addressed in a revised document.

Minor concerns:
1) Growth rate comparisons (i.e.lines 255-263): How does increased culture alkalinity due to ammonia production affect growth?How can you be certain that A1 grows slower in vivo, not just in vitro, especially considering the relatively high starting inoculum for the curves (10^6)?
2) Lines 329-330: As mentioned later in the manuscript, it can't be said that these are isolates of the same strain.
3) Lines 345-348: It would be worth mentioning that increased urease production has also been found to be associated with longer intracellular persistence (https://doi.org/10.1371/journal.ppat.1007144)4) Lines 361-364: This statement would be best positioned immediately following line 336.5) Lines 376-380: It would be worth mentioning that urease inhibition has also been found to be effective in mice and sheep in the context of Ureaplasma infection.6) Figure 1: Was a scatter of NH3 vs Urea also performed?Suggestions: 1) M13 Molecular Typing: It would be very valuable to sequence the urease gene of the different isolates for comparison (eg SNP genotyping), especially for the patient A isolates.Combining with qRT-PCR of Urease gene products would also be interesting.Could lend some weights to the mutability statements by showing variabiliy in the urease itsefl and/or its production.

Reviewer #1 (Comments for the Author):
The paper by Baker et al. evaluated the association of hyperammonemia, kidney failure and cryptococcosis.They found that patients with renal failure and cryptococcosis had a much higher risk of hyperammonemia, particularly those patients with disseminated cryptococcosis (presence of cryptococcal cells in the bloodstream).Although small, this retrospective study is well conducted and well analyzed.Statistic is appropriate.The results are well illustrated, well presented, and well discussed.The methods are clearly outlined.Few issues are below.

It is unclear whether the isolates from the 29 patients were Cryptococcus neoformans or Cryptococcus gattii.
Thank you for noting this oversight.Only the isolate from one of the 29 patients was Cryotococcus gattii.This one patient had an isolated CNS infection (blood culture negative), a normal peak plasma ammonia level of 33µmol/L and an acute kidney injury (AKI) stage zero .A positive Cryptococcus gattii infection was an inclusion criteria in our study design because it is also a urease producer.https://doi.org/10.1111/febs.13229.We have included the following statement in the methods section: "Our medical record review revealed 29 patients with cryptococcal infection, comprising 28 cases of Cryptococcus neoformans and 1 case of Cryptococcus gattii." We have also edited the Figure S5 to reflect this breakdown.

It is unclear why only few clinical cryptococcal isolates were characterized for urease activity and other fungal characteristics. After all, the authors should have access to all isolates from the 29 patients (either from blood or/and CSF).
Thank you for the important question.Cryptococcus infections between 2014 and 2022 were included in our study.However, the Emory ICMC biorepository was founded in 2016 and only began collecting blood stream isolates in 2018.Additionally, the repository focuses on bacterial blood stream isolates (not CSF) and is not geared toward systemic collection of cryptococcus.When yeast is found on work up of blood cultures it is usually transferred over to the mycology section of the lab.On occasion, blood culture plate may grow yeast which is sent to ICMC though an established workflow and may account for the availability of isolates from 3 patients in the study.Hence our retrospective collection of cryptococcus would not have included all cryptococcus isolates (even those from blood).
The methods sections has been edited to make this more clear: "The Emory Investigational Clinical Microbiological Core (ICMC) biorepository which is an IRB approved biorepository (STUDY00093057) was queried for cryptococcal isolates from patients included in our study.Of the 29 patients, Cryptococcal cells from 3 patients were available and recoverable for analysis and therefore representing a convenience sample of available isolates."

Reviewer #2 (Comments for the Author):
In this study, the authors investigated the risk factors for hyperammonemia in patients with Cryptococcus neoformans and gattii infections.A retrospective analysis of 29 patients revealed that 37.9% had hyperammonemia, with renal failure showing a significant association, increasing the risk four-fold.While disseminated cryptococcal infections had a higher risk of hyperammonemia compared to isolated meningitis, a unique strain with increased urease activity was identified in one patient with extreme hyperammonemia.Urease inhibition with an FDA-approved inhibitor was able to damper urease activity and proliferation by all strains.This manuscript is well written and and the experiments have been thoroughly planned and executed.The subject matter sheds light on an underappreciated complication that should garner strong general interest for the spectrum reader base.The most major limitation, being a low sample size, has been addressed.I only have minor concerns and suggestions, listed below, that should be addressed in a revised document.

Minor concerns:
1) Growth rate comparisons (i.e.lines 255-263): How does increased culture alkalinity due to ammonia production affect growth?How can you be certain that A1 grows slower in vivo, not just in vitro, especially considering the relatively high starting inoculum for the curves (10^6)?
Increased culture alkalinity due to ammonia production was shown to inhibit growth of C. neoformans at pH values of 8.5 or higher (Baker and Casadevall, 2023) but in the experiments conducted in the current study, the pH of cultures in the presence or absence of AHA remained at or below neutral pH.We cannot be certain that the A1 isolate would also grow more slowly in vivo, since previous studies showed slower growth of urease-deficient compared to urease-positive C. neoformans in vitro but faster replication for the urease deletion strain inside macrophages (Fu et al., 2018).
The more pertinent observation in this study is that growth of WT and all patient isolates were inhibited to comparably low levels in the presence of AHA as this is expected to prove helpful in the clinical setting.The following statement has been added to the results: "The growth inhibitory effect of AHA is consistent with previous reports of decreased in vitro growth rates for urease-deficient C. neoformans" 2) Lines 329-330: As mentioned later in the manuscript, it can't be said that these are isolates of the same strain.
We thank the reviewer for suggesting this clarification.The sentence has been edited to read as follows: "Interestingly, subsequent isolates from this patient obtained on days 3 , 5 and 12 on antifungal therapy revealed a reduction in urease activity." 3) Lines 345-348: It would be worth mentioning that increased urease production has also been found to be associated with longer intracellular persistence (https://doi.org/10.1371/journal.ppat.1007144)Thank you for the insightful suggestion.We have edited the sentence to read: "For instance, faster growth rates are associated with increased incidence of lytic release from phagocytes while slower intracellular growth rates, that have been correlated with increased urease activity, prolong the persistence of cryptococcal cells within the phagosome."4) Lines 361-364: This statement would be best positioned immediately following line 336.
We are grateful for the suggestion and have edited the fourth paragraph of the discussion to incorporate the statement originally from Lines 361-364.5) Lines 376-380: It would be worth mentioning that urease inhibition has also been found to be effective in mice and sheep in the context of Ureaplasma infection.
Thank you for this suggestion.We added the following statement in line 380 to 383: "In the context of Ureaplasma infections, the use of a urease inhibitor has been demonstrated to limit the growth of Ureaplasma species in vitro and in vivo in mice and sheep (25,26), and to resolve Ureaplasma-induced hyperammonemia in mice( 27)." 6) Figure 1: Was a scatter of NH3 vs Urea also performed?Thank you for this suggestion.We chose to focus on creatinine to correlate renal dysfunction as a risk factor for hyperammonemia because BUN would be a substrate for the urease enzyme and may not reflect the level of renal failure.We have provided below the requested NH3 vs. BUN scatter plot.There is the possibility that a low urea: creatinine ratio may predict a urease producing infection however that was not clearly reflected in our data.This may be confounded by multiple factors: 1. Rate of renal failure may outpace urease activity 2. Variability in urease activity with each strain 3. Nutritional state of the patient as BUN may be elevated in a catabolic state or anabolic resistance during sepsis.4. The small sample size of our study especially pertaining to patient with moderate to severe hyperammonemia (i.e.> 100 µmol/L).We have added the NH3 vs. Urea scatter plot as supplementary data labeled : Figure S6.We thank the reviewer for these insightful suggestions.We have completed DNA sequence analysis of the urease open reading frame and quantitative real-time PCR to compare urease expression in WT and the patient isolates.The sequence data have been added to supplementary Figure S1 and the expression analyses have been added as Figure 5 in the revised manuscript.This analysis has improved the manuscript as it revealed that increased ammonia production by the hyperammonemia patient isolates was not due to overproduction of urease.We also extended our analysis to compare intracellular urease activity which, in agreement with the expression data, showed lower activity for patient isolate A1 compared to WT.These observations suggest that increased ammonia production by the hyperammonemia patient isolate is due to increased extracellular transport of urease.Reviewers determined the manuscript was acceptable Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Sincerely, Kirsten Nielsen Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): The authors responded to my comments in a satisfactory manner.
Reviewer #2 (Comments for the Author): I have no further comments.