In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Increased Alternative Complement Pathway Function and Improved Survival during Critical Illness.

Rationale: Complement is crucial for host defense but may also drive dysregulated inflammation. There is limited understanding of alternative complement function, which can amplify all complement activity, during critical illness.Objectives: We examined the function and key components of the alternative complement pathway in a series of critically ill patients and in a mouse pneumonia model.Methods: Total classical (CH50) and alternative complement (AH50) function were quantified in serum from 321 prospectively enrolled critically ill patients and compared with clinical outcomes. Alternative pathway (AP) regulatory factors were quantified by ELISA (n = 181) and examined via transcriptomics data from external cohorts. Wild-type, Cfb-/-, and C3-/- mice were infected intratracheally with Klebsiella pneumoniae (KP) and assessed for extrapulmonary dissemination.Measurements and Main Results: AH50 greater than or equal to median, but not CH50 greater than or equal to median, was associated with decreased 30-day mortality (adjusted odds ratio [OR], 0.53 [95% confidence interval (CI), 0.31-0.91]), independent of chronic liver disease. One-year survival was improved in patients with AH50 greater than or equal to median (adjusted hazard ratio = 0.59 [95% CI, 0.41-0.87]). Patients with elevated AH50 had increased levels of AP factors B, H, and properdin, and fewer showed a "hyperinflammatory" subphenotype (OR, 0.30 [95% CI, 0.18-0.49]). Increased expression of proximal AP genes was associated with improved survival in two external cohorts. AH50 greater than or equal to median was associated with fewer bloodstream infections (OR, 0.67 [95% CI, 0.45-0.98). Conversely, depletion of AP factors, or AH50 less than median, impaired in vitro serum control of KP that was restored by adding healthy serum. Cfb-/- mice demonstrated increased extrapulmonary dissemination and serum inflammatory markers after intratracheal KP infection compared with wild type.Conclusions: Elevated AP function is associated with improved survival during critical illness, possibly because of enhanced immune capacity.

Functional trait thermal acclimation differs across three species of mid-Atlantic harmful algae.

Characterizing the thermal niche of harmful algae is crucial for understanding and projecting the effects of future climate change on harmful algal blooms. The effects of 6 different temperatures (18-32 °C) on the growth, photophysiology, and toxicity were examined in the dinoflagellate Karlodinium veneficum, and the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa from the Delaware Inland Bays (DIB). K. veneficum and H. akashiwo had skewed unimodal growth patterns, with temperature optima (Topt) at 28.6 and 27.3 °C respectively and an upper thermal niche limit of 32 °C. In contrast, C. subsalsa growth increased linearly with temperature, suggesting Topt and upper thermal boundaries >32 °C. K. veneficum photosystem II (PSII) photochemical efficiency remained stable across all temperatures, while H. akashiwo PSII efficiency declined at higher temperature and C. subsalsa was susceptible to low temperature (~18 °C) photoinactivation. Cell toxicity thermal response was species-specific such that K. veneficum toxicity increased with temperature above Topt. Raphidophyte toxicity peaked at 25-28 °C and was in close agreement with Topt for growth in H. akashiwo but below C. subsalsa maximal growth. The mode of toxicity was markedly different between the dinoflagellate and the raphidophytes such that K. veneficum had greater hemolytic activity while the raphidophytes had pronounced fish gill cell toxicity. These results and patterns of natural abundance for these algae in the DIB suggest that continued ocean warming may contribute to C. subsalsa bloom formation while possibly promoting highly toxic blooms of K. veneficum.